Michael F Brown

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Courses

Scholarly Contributions

Chapters

• Struts, A. V., & Brown, M. F. (2014). Structural dynamics of retinal in rhodopsin activation viewed by solid-state 2H NMR spectroscopy. In Advances in Biological Solid-State NMR: Proteins and Membrane-Active Peptides(pp 320-352).
• Brown, M. F., & Chan, S. I. (2007). Bilayer Membranes: Deuterium and Carbon-13 NMR. In Encyclopedia of Magnetic Resonance. Chichester: John Wiley & Sons.
• Brown, M. F., Lope-Piedrafita, S., Martinez, G. V., & Petrache, H. I. (2006). Solid-State Deuterium NMR Spectroscopy of Membranes. In Modern Magnetic Resonance(pp 245-256). Heidelberg: Springer.
• Brown, M. F. (1996). Membrane Structure and Dynamics Investigated with NMR Spectroscopy. In Biological Membranes: A Molecular Perspective from Computation and Experiment(pp 175-252). Basel: Birkhäuser.
• Brown, M. F., & Chan, S. I. (1996). Bilayer Membranes: Deuterium & Carbon-13 NMR. In Encyclopedia of Nuclear Magnetic Resonance(pp 871-885).
• Brown, M. F., & Gibson, N. J. (1992). Biological Function of Docosahexaenoic Acid in the Retinal Rod Disk Membrane. In Essential Fatty Acids and Eicosanoids(pp 134-138). Champaign, Illinois: American Oil Chemist's Society Press.
• Brown, M. F., Dodd, S. W., & Salmon, A. (1989). Deuterium NMR Spectroscopy of Saturated and Polyunsaturated Lipid Bilayers. In Highlights of Modern Biochemistry(pp 725-734). Zeist: VSP International.
• Trindle, C., Brown, M. F., & Newton, M. G. (1984). Use of Algebraic Symbol-Manipulation Programs in Chemical Research and Education. In Computer Education of Chemists(pp 93-107).
• Fleischer, S., Wang, C. T., Hymel, L., Seelig, J., Brown, M. F., Herbette, L., Scarpa, A., McLaughlin, A. C., & Blasie, J. K. (1979). Structural Studies of the Sarcoplasmic Reticulum Membrane Using the Reconstitution Approach. In Function and Molecular Aspects of Biomembrane Transport(pp 465-485). Amsterdam: Elsevier/North-Holland.

Journals/Publications

• Brown, M. F., Chakraborty, S., & Doktorova, M. (2020). How Cholesterol Stiffens Unsaturated Lipid Membranes. Proc. Natl. Acad. Sci. U.S.A., 117, 21896-21905.
• Brown, M. F., Molugu, T. V., Oita, R. A., Chawla, U., Camp, S. M., & Garcia, J. G. (2020). Nicotinamide Phosphoribosyltransferase Purification Using SUMO Expression System. Anal. Biochem., 598, 113597.
• Brown, M. F., Perera, S. M., Xu, X., Molugu, T. R., & Struts, A. V. (2020). Rhodopsin Activation in Lipid Membranes Based on Solid-State NMR Spectroscopy. Encyclopedia of Biophysics.
• Brown, M. F. (2019). Cholesterol effects on the physical properties of lipid membranes viewed by solid-state NMR spectroscopy. Advances in Experimental Medicine and Biology.
• Brown, M. F. (2019). Collective Dynamics in Lipid Membranes. Characterization of Biological Membranes. Structure and Dynamics.
• Brown, M. F. (2019). Deuterium NMR Spectroscopy in Colloid and Surface Chemistry. Abstr. Pap. Am. Chem. Soc.
• Brown, M. F. (2019). Flexible Lipid Nanomaterials Studied by NMR Spectroscopy. Phys. Chem. Chem. Phys..
• Brown, M. F. (2019). Flexible Surface Model for Lipid-Protein Interactions. Abstr. Pap. Am. Chem. Soc.
• Brown, M. F. (2019). Quantum Mechanical and Molecular Mechanics Modeling of Membrane-Embedded Rhodopsin. J. Membr. Biol.. doi:10.1007/s00232-019-00095-0
• Brown, M. F. (2019). Quasielastic and Elastic Neutron Scattering of Membrane Proteins. Abstr. Pap. Am. Chem. Soc.
• Brown, M. F. (2019). Soft Interfaces in Membrane Protein-Lipid Interactions. Abstr. Pap. Am. Chem. Soc.
• Brown, M. F. (2019). Total synthesis of 9-CD3-9-cis-retinal for studying vision. Abstr. Pap. Am. Chem. Soc.
• Salinas, A. M., Perera, S. M., & Brown, M. F. (2019). Effect of Biopolymer Tethers on Antimicrobial Peptide Activity in Biomembranes. Biophysical Journal.
• Salinas, A. M., Perera, S. M., & Brown, M. F. (2019). Functional Water Dynamics in Rhodopsin Using Solid-State Deuterium NMR Spectroscopy. FASEB Journal.
• Salinas, A. M., Perera, S. M., & Brown, M. F. (2019). G-Protein-Coupled Receptor Activation Mediated by Internal Hydration. Biophysical Journal.
• Salinas, A. M., Perera, S. M., & Brown, M. F. (2019). Hydration Modulates G-Protein-Coupled Receptor Signaling. Biophysical Journal.
• Salinas, A. M., Perera, S. M., & Brown, M. F. (2019). Membrane Deformation Under Isotropic External Stress. Biophysical Journal.
• Salinas, A. M., Perera, S. M., & Brown, M. F. (2019). Mesoscopic Dynamics in Phospholipid Membranes under Osmotic Stress. Biophysical Journal.
• Salinas, A. M., Perera, S. M., & Brown, M. F. (2019). Phospholipid Headgroups Govern Emergent Bending Energy of Membranes with Implications for Lipid-Protein Interactions. Biophysical Journal.
• Salinas, A. M., Perera, S. M., & Brown, M. F. (2019). Retinal Flipping During Rhodopsin Activation Revealed by Solid State 2H NMR and QM/MM Simulations. Biophysical Journal.
• Salinas, A. M., Perera, S. M., & Brown, M. F. (2019). Rhodopsin Hydration Dynamics Studied by Solid-State Deuterium NMR Spectroscopy. Biophysical Journal.
• Salinas, A. M., Perera, S. M., & Brown, M. F. (2019). Solid-State 2H NMR Investigations of Viral M2 Ion Channel Drugs. Biophysical Journal.
• Salinas, A. M., Perera, S. M., & Brown, M. F. (2019). Sponge Model of G-Protein Binding and Unbinding in Membranes. Biophysical Journal.
• Salinas, A. M., Perera, S. M., & Brown, M. F. (2019). Structural Fluctuations in Rhodopsin Activation Revealed by Neutron Scattering. Biophysical Journal.
• Salinas, A. M., Perera, S. M., & Brown, M. F. (2019). Study of Ultra-Fast Rhodopsin Activation Dynamics with Molecular Dynamics Simulations. Biophysical Journal.
• Brown, M. F. (2018). Small-Angle Neutron Scattering Reveals Energy Landscape For Rhodopsin Photoactivation. Journal of Physical Chemistry Letters.
• Brown, M. F. (2018). Solid-State 2H NMR Studies of Water-Mediated Lipid Membrane Deformation. Modern Magnetic Resonance.
• Brown, M. F. (2018). Synthesis of 9-CD3-9-cis-Retinal Cofactor of Isorhodopsin. Tetrahedron Letters.
• Brown, M. F., Mertz, B., & Ritter, E. (2018). Soft Matter Influences on G-Protein-Coupled-Receptor Activation Probed by FTIR and UV-Visible Spectroscopy. Biophysical Journal.
• Fried, S. D., Resager, W. C., Perera, S. M., Brown, M. F., & Marty, M. T. (2018). Investigation of Photoinduced Oligomerization of Rhodopsin By Native Mass Spectrometry. Biophysical Journal.
• Kinnun, J. J., Mallikarjunaiah, K. J., Petrache, H. I., & Brown, M. F. (2018). Emergence of Membrane Material Parameters Revealed by Solid-State 2H NMR Spectroscopy. Biophysical Journal.
• Lee, S., Musharrafieh, R., Xu, X., Struts, A. V., Wang, J., Molugu, T. R., & Brown, M. F. (2018). Solid-State 2H NMR Investigations of Viral M2 Ion Channel Drugs. Biophysical Journal.
• Mologu, T. R., Lee, S., Xu, X., Mallikarjunaiah, K. J., Job, C., & Brown, M. F. (2018). Emergence of Undulations as 2-D Director Fluctuations in Phospholipid Membranes. Biophysical Journal.
• Weerasinghe, N., Fried, S. D., Perera, S. M., Eitel, A. R., Chawla, U., Molugu, T. R., Struts, A. V., & Brown, M. F. (2018). G-Protein-Coupled Receptor Activation through Membrane Deformation. Biophysical Journal.
• Brown, M. F. (2017). Soft Matter in Lipid-Protein Interactions. Annual Review of Biophysics.
• Brown, M. F., Chawla, U., & Perera, S. M. (2017). Membrane Lipid-Protein Interactions. The Biophysics of Cell Membranes: Biological Consequences.
• Chawla, U., Perera, S. M., Struts, A. V., Pitman, M. C., & Brown, M. F. (2017). Role of Soft Matter in G-Protein-Coupled Receptor Signaling. Biophysical Journal.
• Mologu, T. R., Lee, S. K., Musharrafieh, R., Mallikarjunaiah, K. J., Job, C., & Brown, M. F. (2017). Hydration-Mediated Elastic Deformations in Biological Membranes. Biophysical Journal.
• Molugu, T. R., Chawla, U., Huang, A., Oita, R. C., Wang, T., Brown, M. F., & Garcia, J. G. (2017). Self-Association and Conformational Stability of NAMPT Protein. Biophysical Journal.
• Molugu, T. R., Lee, S., & Brown, M. F. (2017). Concepts and Methods of Deuterium NMR Spectroscopy Applied to Biomembranes. Chemical Reviews.
• Molugu, T. R., Xu, X., Leftin, A., Lee, S. K., Mallikarjunaiah, K. J., & Brown, M. F. (2017). Solid-State 2H NMR Studies of Water-Mediated Lipid Membrane Deformation. Modern Magnetic Resonance.
• Molugu, T. R., Xu, X., Leftin, A., Lope-Piedrafita, S., Martinez, G. V., Petrache, H. I., & Brown, M. F. (2017). Solid-State Deuterium NMR Spectroscopy of Membranes. Modern Magnetic Resonance.
• Perera, S. M., Chawla, U., Struts, A. V., & Brown, M. F. (2017). Energy Landscape and Spatial Motion Models in Rhodopsin Activation. Biophysical Journal.
• Perera, S. M., Xu, X., Mologu, T. R., Mallikarjuniah, K. J., Struts, A. V., & Brown, M. F. (2017). Solid-State Deuterium NMR Spectroscopy of Rhodopsin. Modern Magnetic Resonance.
• Perera, S. M., Xu, X., Struts, A. V., Chawla, U., Boutet, S., Carbajo, S., Seaberg, M. D., Hunter, M. S., Martin-Garcia, J. M., Coe, J. D., Wiedorn, M. O., Nelson, G., Chamberlain, S., Deponte, D. P., Fromme, R., Grant, T. D., Kirian, R. A., Fromme, P., & Brown, M. F. (2017). Time-Resolved Wide-Angle X-ray Scattering Reveals Protein Quake in Rhodopsin Activation. Biophysical Journal.
• Salinas, A. M., Perera, S. M., & Brown, M. F. (2018). Hydration Thermodynamics of a Powdered G-Protein-Coupled Receptor. Biophysical Journal.
• Struts, A. V., Xu, X., Molugu, T. R., Pitman, M. C., Faylough, S., Nascimento, C., Nesnas, N., & Brown, M. F. (2017). Activation of GPCR Rhodopsin Investigated by Solid-State NMR Spectroscopy. Biophysical Journal.
• Xu, X., Struts, A. V., Giri, A. K., Mologu, T. R., Guruge, C., Faylough, S., Nascimento, C. L., Nesnas, N., Hruby, V. J., & Brown, M. F. (2018). Dynamics of Membrane Proteins Studied by Solid State 2H NMR Relaxation. Biophysical Journal.
• Xu, X., Struts, A. V., Mologu, T. R., Perera, S. M., Chawla, U., Lee, S. K., Musharrafieh, R., Johnson, A. M., Huang, A., Knowles, T. A., & Brown, M. F. (2017). Multiscale GPCR Activation in Lipid Membranes Probed by Solid-State NMR and Scattering Methods. Biophysical Journal.
• Brown, M. F., & Molugu, T. R. (2016). Cholesterol-Induced Suppression of Membrane Elastic Fluctuations at the Atomistic Level. Chemistry and Physics of Lipids, 199, 39-51.
• Chawla, U., Jiang, Y., Zheng, W., Perera, S. M., Brown, M. F., & Liang, H. (2016). A Usual G-Protein-Coupled Receptor in Unusual Membranes. Angewandte Chemie, 128, 598-602. doi:DOI: 10.1002/ange.201508648
• Chawla, U., Perera, S. M., Struts, A. V., Pitman, M. C., & Brown, M. F. (2016). Hydration Mediated G-Protein-Coupled Receptor Activation,. Biophysical Journal, 110, 83a.
• Lee, S. K., Molugu, T. R., Brown, M. F., & Mallikarjunaiah, K. J. (2016). Elastic Deformation and Collective Dynamics in Lipid Membranes: A Solid-State 2H NMR Relaxation Study. Biophysical Journal, 110, 396a.
• Molugu, T. R., Chawla, U., Oita, R. C., Wang, T., Brown, M. F., & Garcia, J. G. (2016). Thermal Denaturation and Domain Stability of NAMPT Protein. Biophysical Journal, 110, 44a.
• Musharrafieh, R., Chawla, U., Zheng, W., Kaung, L., Perera, S. M., Knowles, T., Huang, A., Pitman, M. C., Wang, J., Liang, H., & Brown, M. F. (2016). Computational and Experimental Studies of Lipid-Protein Interactions in Biomemrane Function. Biophysical Journal, 110, 257a.
• Perera, S. M., Chawla, U., & Brown, M. F. (2016). Powdered G-Protein–Coupled Receptors. Journal of Physical Chemistry Letters, 7, 4230-4235.
• Perera, S. M., Shresta, U., Bhowmik, D., Struts, A. V., Chu, X., & Brown, M. F. (2016). Neutron Scattering Reveals Protein Fluctuations in GPCR Activation. Biophysical Journal, 110, 228a-229a.
• Shrestha, U. R., Perera, S. M., Bhowmik, D., Chawla, U., Mamontov, E., Brown, M. F., & Chu, X. (2016). Quasi-Elastic Neutron Scattering Reveals Ligand-Induced Protein Dynamics of a G-Protein–Coupled Receptor. Journal of Physical Chemistry Letters, 7, 4130−4136.
• Struts, A. V., Barmasov, A. V., & Brown, M. F. (2016). Spectral Methods for Study of the G-Protein-Coupled Receptor Rhodopsin. II. Magnetic Resonance Spectroscopy. Optics and Spectroscopy, 118, 711-717.
• Struts, A. V., Xu, X., Molugu, T. R., Pitman, M. C., Faylough, S., Guruge, C., Nascimento, C., Nesnas, N., & Brown, M. F. (2016). Retinal Chromophore Structure in Meta-II Rhodopsin Revealed by Solid-State 2H NMR and Molecular Modeling. Biophysical Journal, 110, 229a-230a.
• Bhowmik, D., Shrestha, U., Perera, S. M., Chawla, U., Mamontov, E., Brown, M. F., & Chu, X. Q. (2015). Rhodopsin Photoactivation Dynamics Revealed by Quasi-Elastic Neutron Scattering. Biophysical Journal, 108, 61a.
• Chawla, U., Jiang, Y., Zheng, W., Perera, S. M., Brown, M. F., & Liang, H. (2015). A Usual G-Protein-Coupled Receptor in Unusual Membranes. Angewandte Chemie International Edition, 54, 1–6. doi:DOI: 10.1002/anie.201508648
• Chawla, U., Mertz, B., Ritter, E., Bartl, F., & Brown, M. F. (2015). Membrane-Lipid Mediated Rhodopsin Signaling Involves an Ensemble of Conformational Substates. Biophysical Journal, 108, 556a.
• Chawla, U., Zheng, W., Kuang, L., Jiang, Y., Perera, S. M., Brown, M. F., & Liang, H. (2015). Spontaneous Reconstitution of Bovine Rhodopsin into Artificial Membranes. Biophysical Journal, 108, 500a-501a.
• Feng, J., Mertz, B., & Brown, M. F. (2015). Retinal Flip in Rhodopsin Activation?. Biophysical Journal, 108, 2767-2770.
• Kinnun, J. J., Mallikarjunaiah, K. J., Petrache, H. I., & Brown, M. F. (2015). Elastic Deformation and Area Per Lipid of Membranes: Atomistic View From Solid-State Deuterium NMR Spectroscopy. Biochimica et Biophysica Acta, 1848, 246-259.
• Molugu, T. R., Lee, S. K., Job, C., & Brown, M. F. (2015). Hydration-Modulated Collective Dynamics of Membrane Lipids are Revealed by Solid-State 2H NMR Relaxation. Biophysical Journal, 108, 77a.
• Shrestha, U., Bhowmik, D., Perera, S. M., Chawla, U., Struts, A. V., Graziano, V., Qian, S., Heller, W. T., Brown, M. F., & Chu, X. Q. (2015). Small Angle Neutron and X-Ray Scattering Reveal Conformational Differences in Detergents Affecting Rhodopsin Activation. Biophysical Journal, 108, 39a.
• Struts, A. V., Barmasov, A. V., & Brown, M. F. (2015). Spectral Methods for Study of the G-Protein-Coupled Receptor Rhodopsin. I. Vibrational and Electronic Spectroscopy. Optics and Spectroscopy, 711-717.
• Struts, A. V., Chawla, U., Perera, S. M., & Brown, M. F. (2015). Investigation of Rhodopsin Dynamics in its Signaling State by Solid-State Deuterium NMR Spectroscopy. Methods in Molecular Biology, 1271, 133–158.
• Xu, X., Struts, A. V., Giri, A. K., Molugu, T. R., Guruge, C., Faylough, S., Nascimento, C. L., Nesnas, N., Hruby, V. J., & Brown, M. F. (2015). Solid-State 2H NMR Investigation of Transducin Activation by Rhodopsin. Biophysical Journal, 108, 411a.
• Brown, M. F. (2014). Conformational Fluctuations in G-Protein-Coupled Receptors. Bulletin of the American Physical Society, 59.
• Brown, M. F., Chawla, U., Perera, S. M., & Struts, A. V. (2014). Lipid-Mediated Activation of G-Protein-Coupled Receptors in Membranes. Bulletin of the American Physical Society, 59.
• Brown, M. F., Chawla, U., Perera, S. M., & Struts, A. V. (2014). Role of Membrane Lipids in Activating G-Protein-Coupled Receptors. Biophysical Journal, 106, 434a.
• Brown, M. F., Perera, S. M., Shrestha, U., Chawla, U., Struts, A. V., Qian, S., Brown, M. F., & Chu, X. Q. (2014). G-Protein-Coupled Receptor Activation Investigated using Small-Angle Neutron Scattering. Biophysical Journal, 106, 634a.
• Brown, M. F., Xu, X., & Struts, A. V. (2014). Generalized Model-Free Analysis of Nuclear Spin Relaxation Experiments. Encyclopedia of Magnetic Resonance, 3, 275–286.
• Chawla, U., Mertz, B., Ritter, E., Bartl, F., & Brown, M. F. (2014). Investigation of Lipid Bilayer Effects on Rhodopsin Activation using UV-Visible and FTIR Spectroscopy. Biophysical Journal, 106, 715a.
• Chu, X. Q., Perera, S., Shrestha, U., Chawla, U., Struts, A. V., Qian, S., & Brown, M. F. (2014). Ensemble Activation of G-Protein–Coupled Receptors Revealed by Small-Angle Neutron Scattering. Bulletin of the American Physical Society, 59.
• Leftin, A., Molugu, T. R., Job, C., Beyer, K., & Brown, M. F. (2014). Area per lipid and cholesterol interactions in membranes from separated local-field 13C NMR spectroscopy. Biophysical Journal, 107, 2274–2286.
• Leftin, A., Xu, X., & Brown, M. F. (2014). Phospholipid Bilayer Membranes: Deuterium and Carbon-13 NMR Spectroscopy. Encyclopedia of Magnetic Resonance, 3, 199–214.
• Leioatts, N., Mertz, B., Martínez-Mayorga, K., Romo, T. D., Pitman, M. C., Feller, S. E., Grossfield, A., & Brown, M. F. (2014). Retinal Makes Concerted Conformational Changes During Early Stages of Rhodopsin Activation. Biophysical Journal, 106, 54a.
• Leioatts, N., Mertz, B., Martínez-Mayorga, K., Romo, T. D., Pitman, M. C., Feller, S. E., Grossfield, A., & Brown, M. F. (2014). Retinal ligand mobility explains internal hydration and reconciles active rhodopsin structures. Biochemistry, 53(2), 376-385.
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  Abstract: Rhodopsin, the mammalian dim-light receptor, is one of the best-characterized G-protein-coupled receptors, a pharmaceutically important class of membrane proteins that has garnered a great deal of attention because of the recent availability of structural information. Yet the mechanism of rhodopsin activation is not fully understood. Here, we use microsecond-scale all-atom molecular dynamics simulations, validated by solid-state 2H nuclear magnetic resonance spectroscopy, to understand the transition between the dark and metarhodopsin I (Meta I) states. Our analysis of these simulations reveals striking differences in ligand flexibility between the two states. Retinal is much more dynamic in Meta I, adopting an elongated conformation similar to that seen in the recent activelike crystal structures. Surprisingly, this elongation corresponds to both a dramatic influx of bulk water into the hydrophobic core of the protein and a concerted transition in the highly conserved Trp2656.48 residue. In addition, enhanced ligand flexibility upon light activation provides an explanation for the different retinal orientations observed in X-ray crystal structures of active rhodopsin. © 2013 American Chemical Society.
• Mallikarjunaiah, K. J., Feng, J., Mertz, B., & Brown, M. F. (2014). Experimental and Theoretical Comparison of Pressure Effects on Lipid Bilayer Fluctuations. Biophysical Journal, 106, 80a.
• Mallikarjunaiah, K. J., Palacio, L. A., Petrache, H. I., Kinnun, J. J., & Brown, M. F. (2014). Intermembrane Forces and Membrane Deformation Observed via Dehydration and Osmotic Pressure. Biophysical Journal, 106, 287a.
• Molugu, T. R., Leftin, A., Job, C., & Brown, M. F. (2014). Area Per Lipid of Membranes from Natural Abundance Solid-State 13C NMR Spectroscopy. Biophysical Journal, 106, 452a.
• Xu, X., Struts, A. V., Mallikarjunaiah, K. J., & Brown, M. F. (2014). Generalized Model-Free Spectral Density Analysis Applied to Rhodopsin Activation in Membranes. Biophysical Journal, 106, 651a-652a.
• Brown, M. F., & Struts, A. V. (2013). Activation of rhodopsin based on solid-state NMR spectroscopy. Encyclopedia of Biophysics, 2231–2243.
• Brown, M. F., Chawla, U., & Perera, S. M. (2013). Membrane Bilayer Environment Influences Thermodynamics of Rhodopsin Membrane Protein-Lipid Interactions. Biophysical Journal, 104, 434.
• Kinnun, J. J., Leftin, A., & Brown, M. F. (2013). Solid-state NMR spectroscopy for the physical chemistry laboratory. Journal of Chemical Education, 90(1), 123-128.
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  Abstract: Solid-state nuclear magnetic resonance (NMR) spectroscopy finds growing application to inorganic and organic materials, biological samples, polymers, proteins, and cellular membranes. However, this technique is often neither included in laboratory curricula nor typically covered in undergraduate courses. On the other hand, spectroscopy and molecular structure taught in second-semester undergraduate physical chemistry courses meet the minimal background prerequisites for interpreting data obtained in many solid-state NMR experiments. A solid-state 2H NMR experiment is described in which the student obtains and interprets the spectrum of a powder sample of hexamethylbenzene-d18 using a solution NMR spectrometer as found in many undergraduate institutions. A quadrupolar-echo pulse sequence is applied to the sample to obtain the 2H NMR spectrum. The spectrum of a randomly oriented powder sample consists of two spectral branches with broad shoulders. The quadrupolar frequencies corresponding to the nuclear spin transitions are interpreted in terms of molecular mobility in the solid state, that is, 3-fold rotation of the methyl groups and 6-fold rotation of the hexamethylbenzene ring. In this way, students discover that solid substances may have significant molecular motions. This undergraduate investigation employing solid-state 2H NMR provides an informative exposure to state-of-the-art research techniques by using facilities that are already in place at many undergraduate institutions. Furthermore, it demonstrates a real-life manifestation of quantum mechanics as explained in physical chemistry courses, as well as fundamentals of molecular motions such as rotation of aromatic ring compounds and methyl groups in the solid state. © 2012 The American Chemical Society and Division of Chemical Education, Inc.
• Kinnun, J. J., Mallikarjunaiah, K. J., Palacio, L. A., Brown, M. F., & Petrache, H. I. (2013). Membrane Structure and Intermembrane Forces Observed with Small Angle X-Ray Scattering. Biophysical Journal, 104, 81.
• Leftin, A., Job, C., Beyer, K., & Brown, M. F. (2013). Solid-State 13C NMR reveals annealing of raft-like membranes containing cholesterol by the intrinsically disordered protein α-synuclein. Journal of Molecular Biology, 425(16), 2973-2987.
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  PMID: 23583776;Abstract: Misfolding and aggregation of the intrinsically disordered protein α-Synuclein (αS) in Lewy body plaques are characteristic markers of late-stage Parkinson's disease. It is well established that membrane binding is initiated at the N-terminus of the protein and affects biasing of conformational ensembles of αS. However, little is understood about the effect of αS on the membrane lipid bilayer. One hypothesis is that intrinsically disordered αS alters the structural properties of the membrane, thereby stabilizing the bilayer against fusion. Here, we used two-dimensional 13C separated local-field NMR to study interaction of the wild-type α-Synuclein (wt-αS) or its N-terminal (1-25) amino acid sequence (N-αS) with a cholesterol-enriched ternary membrane system. This lipid bilayer mimics cellular raft-like domains in the brain that are proposed to be involved in neuronal membrane fusion. The two-dimensional dipolar-recoupling pulse sequence DROSS (dipolar recoupling on-axis with scaling and shape preservation) was implemented to measure isotropic 13C chemical shifts and 13C-1H residual dipolar couplings under magic-angle spinning. Site-specific changes in NMR chemical shifts and segmental order parameters indicate that both wt-αS and N-αS bind to the membrane interface and change lipid packing within raft-like membranes. Mean-torque modeling of 13C-1H NMR order parameters shows that αS induces a remarkable thinning of the bilayer (≈ 6 Å), accompanied by an increase in phospholipid cross-sectional area (≈ 10 Å2). This perturbation is characterized as membrane annealing and entails structural remodeling of the raft-like liquid-ordered phase. We propose this process is implicated in regulation of synaptic membrane fusion that may be altered by aggregation of αS in Parkinson's disease. © 2013 Elsevier Ltd.
• Leioatts, N., Mertz, B., Martínez-Mayorga, K., Romo, T. D., Pitman, M. C., Feller, S. E., Grossfield, A., & Brown, M. F. (2013). Retinal Changes Conformation During the Early Stages of Rhodopsin Activation. Biophysical Journal, 104, 114.
• Mallikarjunaiah, K. J., Kinnun, J. J., Petrache, H. I., & Brown, M. F. (2013). Area per Lipid and Elastic Deformation of Membrane Bilayers Under Osmotic Stress. Biophysical Journal, 104, 588.
• Mallikarjunaiah, K. J., Zheng, M., Myers, E. M., Kinnun, J. J., Petrache, H. I., & Brown, M. F. (2013). Restructuring of Membrane Bilayers due to Osmotic Pressure: Deuterium Solid-State NMR Study. Biophysical Journal, 104, 33.
• Mertz, B., Ritter, E., Bartl, F., & Brown, M. F. (2013). Lipid Bilayer Influences Rhodopsin Activation Probed by FTIR and UV-Visible Spectroscopy. Biophysical Journal, 104, 364.
• Molugu, T. R., Mallikarjunaiah, K. J., Job, C., & Brown, M. F. (2013). Suppression of Cooperative Motions in Phospholipid Membranes by Osmotic Stress: Deuterium NMR Relaxation Study. Biophysical Journal, 104, 81.
• Struts, A. V., Xu, X., Mallikarjunaiah, K. J., & Brown, M. F. (2013). Model-Free Spectral Density Mapping Applied to Dynamics of Rhodopsin in Solid-State NMR Spectroscopy. Biophysical Journal, 104, 41.
• Zhu, S., Brown, M. F., & Feller, S. (2013). Retinal Conformation Governs pKa of Protonated Schiff Base in Rhodopsin Activation. Biophysical Journal, 104, 114.
• Zhu, S., Brown, M. F., & Feller, S. E. (2013). Retinal conformation governs pKa of protonated schiff base in rhodopsin activation. Journal of the American Chemical Society, 135(25), 9391-9398.
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  PMID: 23701524;Abstract: We have explored the relationship between conformational energetics and the protonation state of the Schiff base in retinal, the covalently bound ligand responsible for activating the G protein-coupled receptor rhodopsin, using quantum chemical calculations. Guided by experimental structural determinations and large-scale molecular simulations on this system, we examined rotation about each bond in the retinal polyene chain, for both the protonated and deprotonated states that represent the dark and photoactivated states, respectively. Particular attention was paid to the torsional degrees of freedom that determine the shape of the molecule, and hence its interactions with the protein binding pocket. While most torsional degrees of freedom in retinal are characterized by large energetic barriers that minimize structural fluctuations under physiological temperatures, the C6-C7 dihedral defining the relative orientation of the β-ionone ring to the polyene chain has both modest barrier heights and a torsional energy surface that changes dramatically with protonation of the Schiff base. This surprising coupling between conformational degrees of freedom and protonation state is further quantified by calculations of the pKa as a function of the C6-C7 dihedral angle. Notably, pKa shifts of greater than two units arise from torsional fluctuations observed in molecular dynamics simulations of the full ligand-protein-membrane system. It follows that fluctuations in the protonation state of the Schiff base occur prior to forming the activated MII state. These new results shed light on important mechanistic aspects of retinal conformational changes that are involved in the activation of rhodopsin in the visual process. © 2013 American Chemical Society.
• Zook, J. D., Molugu, T. D., Jacobsen, N. E., Lin, G., Soll, J., Cherry, B. R., Brown, M. F., & Fromme, P. (2013). High-resolution NMR reveals secondary structure and folding of amino acid transporter from outer chloroplast membrane. PLoS ONE, 8, e78116–e78116.
• Zook, J., Moulgu, T. R., Jacobsen, N., Lin, G., Cherry, B., Brown, M. F., & Fromme, P. (2013). Secondary Structure and Folding of Amino Acid Transporter from Outer Chloroplast Membrane. Biophysical Journal, 104, 221.
• Brown, M. F. (2012). Curvature Forces in Membrane Lipid-Protein Interactions. Bulletin of the American Physical Society, 57.
• Brown, M. F. (2012). Curvature forces in membrane lipid-protein interactions. Biochemistry, 51(49), 9782-9795.
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  Membrane biochemists are becoming increasingly aware of the role of lipid-protein interactions in diverse cellular functions. This review describes how conformational changes in membrane proteins, involving folding, stability, and membrane shape transitions, potentially involve elastic remodeling of the lipid bilayer. Evidence suggests that membrane lipids affect proteins through interactions of a relatively long-range nature, extending beyond a single annulus of next-neighbor boundary lipids. It is assumed the distance scale of the forces is large compared to the molecular range of action. Application of the theory of elasticity to flexible soft surfaces derives from classical physics and explains the polymorphism of both detergents and membrane phospholipids. A flexible surface model (FSM) describes the balance of curvature and hydrophobic forces in lipid-protein interactions. Chemically nonspecific properties of the lipid bilayer modulate the conformational energetics of membrane proteins. The new biomembrane model challenges the standard model (the fluid mosaic model) found in biochemistry texts. The idea of a curvature force field based on data first introduced for rhodopsin gives a bridge between theory and experiment. Influences of bilayer thickness, nonlamellar-forming lipids, detergents, and osmotic stress are all explained by the FSM. An increased awareness of curvature forces suggests that research will accelerate as structural biology becomes more closely entwined with the physical chemistry of lipids in explaining membrane structure and function.
• Brown, M. F. (2012). Flexible Surface Model for Membrane Lipid-Protein Interactions. Biophysical Journal, 102, 6.
• Brown, M. F. (2012). UV-visible and infrared methods for investigating lipid-rhodopsin membrane interactions. Methods in Molecular Biology, 914, 127–153.
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  We describe experimental UV-visible and Fourier transform infrared (FTIR) spectroscopic methods for characterizing lipid-protein interactions for rhodopsin in a membrane bilayer environment. The combination of FTIR and UV-visible difference spectroscopy is used to monitor the structural and functional changes during rhodopsin activation. Investigations of how membrane lipids stabilize various rhodopsin photoproducts are analogous to mutating the protein in terms of gain or loss of function. Interpretation of the results entails a flexible surface model for explaining membrane lipid-protein interactions through material properties relevant to biological activity.
• Brown, M. F., Mertz, B., Martínez-Mayorga, K., Grossfield, A., Medina-Franco, J. L., Pitman, M. C., & Feller, S. E. (2012). Molecular Simulations Illuminate Rhodopsin Activation Based on New Crystal Structures. Biophysical Journal, 102, 468.
• Brown, M., Brown, M. F., Mertz, B., Struts, A. V., & Feller, S. E. (2012). Molecular simulations and solid-state NMR investigate dynamical structure in rhodopsin activation. Biochimica et Biophysica Acta, 1818(2).
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  Rhodopsin has served as the primary model for studying G protein-coupled receptors (GPCRs)-the largest group in the human genome, and consequently a primary target for pharmaceutical development. Understanding the functions and activation mechanisms of GPCRs has proven to be extraordinarily difficult, as they are part of a complex signaling cascade and reside within the cell membrane. Although X-ray crystallography has recently solved several GPCR structures that may resemble the activated conformation, the dynamics and mechanism of rhodopsin activation continue to remain elusive. Notably solid-state ((2))H NMR spectroscopy provides key information pertinent to how local dynamics of the retinal ligand change during rhodopsin activation. When combined with molecular mechanics simulations of proteolipid membranes, a new paradigm for the rhodopsin activation process emerges. Experiment and simulation both suggest that retinal isomerization initiates the rhodopsin photocascade to yield not a single activated structure, but rather an ensemble of activated conformational states. This article is part of a Special Issue entitled: Membrane protein structure and function.
• Kinnun, J. J., Mallikarjunaiah, K. J., Petrache, H. I., & Brown, M. F. (2012). Intermembrane Forces Probed by Osmotic Stress and Solid-State 2H NMR Spectroscopy. Biophysical Journal, 102, 82.
• Lewis, J. W., Szundi, I., Kliger, D. S., & Brown, M. F. (2012). Time-Resolved UV-Visible Studies of Rhodopsin Provide Experimental Test of Flexible Surface Model for Lipid-Protein Interactions. Biophysical Journal, 102, 627.
• Mallikarjunaiah, K. J., & Brown, M. F. (2012). Solid-State 2H NMR Reveals Changes in Membrane Flexibility Due to Osmotic Pressure. Biophysical Journal, 102, 292.
• Mallikarjunaiah, K. J., Kinnun, J. J., Petrache, H. I., & Brown, M. F. (2012). Membrane Area Deformation under Osmotic Stress: Deuterium NMR Approach. Biophysical Journal, 505.
• Mertz, B., Brown, M. F., & Lyman, E. R. (2012). Rhodopsin Simulations in Detergent Micelles Characterize Meta I and Meta II Photointermediates. Biophysical Journal, 624.
• Mertz, B., Ritter, E., Bartl, F., & Brown, M. F. (2012). Rhodopsin Activation is Modulated by Non-Specific Membrane Lipid-Protein Interactions. Biophysical Journal, 102, 468.
• Molugu, T. R., Mallikarjunaiah, K. J., Job, C., & Brown, M. F. (2012). Hydration-Mediated Slow Dynamics in Phospholipid Membranes. Biophysical Journal, 102, 389.
• Olausson, B. E., Grossfield, A., Pitman, M. C., Brown, M. F., Feller, S. E., & Vogel, A. (2012). Molecular dynamics simulations reveal specific interactions of post-translational palmitoyl modifications with rhodopsin in membranes. Journal of the American Chemical Society, 134(9), 4324-4331.
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  PMID: 22280374;PMCID: PMC3299983;Abstract: We present a detailed analysis of the behavior of the highly flexible post-translational lipid modifications of rhodopsin from multiple-microsecond all-atom molecular dynamics simulations. Rhodopsin was studied in a realistic membrane environment that includes cholesterol, as well as saturated and polyunsaturated lipids with phosphocholine and phosphoethanolamine headgroups. The simulation reveals striking differences between the palmitoylations at Cys322 and Cys323 as well as between the palmitoyl chains and the neighboring lipids. Notably the palmitoyl group at Cys322 shows considerably greater contact with helix H1 of rhodopsin, yielding frequent chain upturns with longer reorientational correlation times, and relatively low order parameters. While the palmitoylation at Cys323 makes fewer protein contacts and has increased order compared to Cys322, it nevertheless exhibits greater flexibility with smaller order parameters than the stearoyl chains of the surrounding lipids. The dynamical structure of the palmitoylations - as well as their extensive fluctuations - suggests a complex function for the post-translational modifications in rhodopsin and potentially other G protein-coupled receptors, going beyond their role as membrane anchoring elements. Rather, we propose that the palmitoylation at Cys323 has a potential role as a lipid anchor, whereas the palmitoyl-protein interaction observed for Cys322 suggests a more specific interaction that affects the stability of the dark state of rhodopsin. © 2012 American Chemical Society.
• Zhu, S., Mertz, B., Brown, M. F., & Feller, S. E. (2012). The Retinal Energy Landscape as a Function of the Rhodopsin Photocycle. Biophysical Journal, 102, 239.
• Zook, J. D., Brown, M. F., Jacobsen, N., Lin, G., & Fromme, P. (2012). NMR Studies on Membrane Protein OEP16. Biophysical Journal, 102, 263.
• Brown, M., Brown, M. F., Mallikarjunaiah, K. J., Leftin, A., Kinnun, J. J., Justice, M. J., Rogozea, A. L., & Petrache, H. I. (2011). Solid-state ²H NMR shows equivalence of dehydration and osmotic pressures in lipid membrane deformation. Biophysical Journal, 100(1).
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  Lipid bilayers represent a fascinating class of biomaterials whose properties are altered by changes in pressure or temperature. Functions of cellular membranes can be affected by nonspecific lipid-protein interactions that depend on bilayer material properties. Here we address the changes in lipid bilayer structure induced by external pressure. Solid-state ²H NMR spectroscopy of phospholipid bilayers under osmotic stress allows structural fluctuations and deformation of membranes to be investigated. We highlight the results from NMR experiments utilizing pressure-based force techniques that control membrane structure and tension. Our ²H NMR results using both dehydration pressure (low water activity) and osmotic pressure (poly(ethylene glycol) as osmolyte) show that the segmental order parameters (S(CD)) of DMPC approach very large values of ≈ 0.35 in the liquid-crystalline state. The two stresses are thermodynamically equivalent, because the change in chemical potential when transferring water from the interlamellar space to the bulk water phase corresponds to the induced pressure. This theoretical equivalence is experimentally revealed by considering the solid-state ²H NMR spectrometer as a virtual osmometer. Moreover, we extend this approach to include the correspondence between osmotic pressure and hydrostatic pressure. Our results establish the magnitude of the pressures that lead to significant bilayer deformation including changes in area per lipid and volumetric bilayer thickness. We find that appreciable bilayer structural changes occur with osmotic pressures in the range of 10-100 atm or lower. This research demonstrates the applicability of solid-state ²H NMR spectroscopy together with bilayer stress techniques for investigating the mechanism of pressure sensitivity of membrane proteins.
• Brown, M., Brown, M. F., Struts, A. V., Salgado, G. F., & Martínez-Mayorga, K. (2011). Retinal dynamics underlie its switch from inverse agonist to agonist during rhodopsin activation. Nature Structural & Molecular Biology, 18(3).
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  X-ray and magnetic resonance approaches, though central to studies of G protein-coupled receptor (GPCR)-mediated signaling, cannot address GPCR protein dynamics or plasticity. Here we show that solid-state (2)H NMR relaxation elucidates picosecond-to-nanosecond-timescale motions of the retinal ligand that influence larger-scale functional dynamics of rhodopsin in membranes. We propose a multiscale activation mechanism whereby retinal initiates collective helix fluctuations in the meta I-meta II equilibrium on the microsecond-to-millisecond timescale.
• Brown, M., Struts, A. V., Salgado, G. F., & Brown, M. F. (2011). Solid-state 2H NMR relaxation illuminates functional dynamics of retinal cofactor in membrane activation of rhodopsin. Proceedings of the National Academy of Sciences of the United States of America, 108(20).
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  Rhodopsin is a canonical member of the family of G protein-coupled receptors, which transmit signals across cellular membranes and are linked to many drug interventions in humans. Here we show that solid-state (2)H NMR relaxation allows investigation of light-induced changes in local ps-ns time scale motions of retinal bound to rhodopsin. Site-specific (2)H labels were introduced into methyl groups of the retinal ligand that are essential to the activation process. We conducted solid-state (2)H NMR relaxation (spin-lattice, T(1Z), and quadrupolar-order, T(1Q)) experiments in the dark, Meta I, and Meta II states of the photoreceptor. Surprisingly, we find the retinylidene methyl groups exhibit site-specific differences in dynamics that change upon light excitation--even more striking, the C9-methyl group is a dynamical hotspot that corresponds to a crucial functional hotspot of rhodopsin. Following 11-cis to trans isomerization, the (2)H NMR data suggest the β-ionone ring remains in its hydrophobic binding pocket in all three states of the protein. We propose a multiscale activation mechanism with a complex energy landscape, whereby the photonic energy is directed against the E2 loop by the C13-methyl group, and toward helices H3 and H5 by the C5-methyl of the β-ionone ring. Changes in retinal structure and dynamics initiate activating fluctuations of transmembrane helices H5 and H6 in the Meta I-Meta II equilibrium of rhodopsin. Our proposals challenge the Standard Model whereby a single light-activated receptor conformation yields the visual response--rather an ensemble of substates is present, due to the entropy gain produced by photolysis of the inhibitory retinal lock.
• Leftin, A., & Brown, M. F. (2011). An NMR database for simulations of membrane dynamics. Biochimica et Biophysica Acta, 1808(3), 818-839.
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  PMID: 21134351;Abstract: Computational methods are powerful in capturing the results of experimental studies in terms of force fields that both explain and predict biological structures. Validation of molecular simulations requires comparison with experimental data to test and confirm computational predictions. Here we report a comprehensive database of NMR results for membrane phospholipids with interpretations intended to be accessible by non-NMR specialists. Experimental 13C-1H and 2H NMR segmental order parameters (SCH or SCD) and spin-lattice (Zeeman) relaxation times (T1Z) are summarized in convenient tabular form for various saturated, unsaturated, and biological membrane phospholipids. Segmental order parameters give direct information about bilayer structural properties, including the area per lipid and volumetric hydrocarbon thickness. In addition, relaxation rates provide complementary information about molecular dynamics. Particular attention is paid to the magnetic field dependence (frequency dispersion) of the NMR relaxation rates in terms of various simplified power laws. Model-free reduction of the T1Z studies in terms of a power-law formalism shows that the relaxation rates for saturated phosphatidylcholines follow a single frequency-dispersive trend within the MHz regime. We show how analytical models can guide the continued development of atomistic and coarse-grained force fields. Our interpretation suggests that lipid diffusion and collective order fluctuations are implicitly governed by the viscoelastic nature of the liquid-crystalline ensemble. Collective bilayer excitations are emergent over mesoscopic length scales that fall between the molecular and bilayer dimensions, and are important for lipid organization and lipid-protein interactions. Future conceptual advances and theoretical reductions will foster understanding of biomembrane structural dynamics through a synergy of NMR measurements and molecular simulations. © 2010 Elsevier B.V. All rights reserved.
• Mallikarjunaiah, K. J., Leftin, A., Kinnun, J. J., Justice, M. J., Rogozea, A. L., Petrache, H. I., & Brown, M. F. (2011). Solid-state 2H NMR shows equivalence of dehydration and osmotic pressures in lipid membrane deformation. Biophysical Journal, 100(1), 98-107.
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  PMID: 21190661;PMCID: PMC3010004;Abstract: Lipid bilayers represent a fascinating class of biomaterials whose properties are altered by changes in pressure or temperature. Functions of cellular membranes can be affected by nonspecific lipid-protein interactions that depend on bilayer material properties. Here we address the changes in lipid bilayer structure induced by external pressure. Solid-state 2H NMR spectroscopy of phospholipid bilayers under osmotic stress allows structural fluctuations and deformation of membranes to be investigated. We highlight the results from NMR experiments utilizing pressure-based force techniques that control membrane structure and tension. Our 2H NMR results using both dehydration pressure (low water activity) and osmotic pressure (poly(ethylene glycol) as osmolyte) show that the segmental order parameters (SCD) of DMPC approach very large values of ≈0.35 in the liquid-crystalline state. The two stresses are thermodynamically equivalent, because the change in chemical potential when transferring water from the interlamellar space to the bulk water phase corresponds to the induced pressure. This theoretical equivalence is experimentally revealed by considering the solid-state 2H NMR spectrometer as a virtual osmometer. Moreover, we extend this approach to include the correspondence between osmotic pressure and hydrostatic pressure. Our results establish the magnitude of the pressures that lead to significant bilayer deformation including changes in area per lipid and volumetric bilayer thickness. We find that appreciable bilayer structural changes occur with osmotic pressures in the range of 10-100 atm or lower. This research demonstrates the applicability of solid-state 2H NMR spectroscopy together with bilayer stress techniques for investigating the mechanism of pressure sensitivity of membrane proteins. © 2011 by the Biophysical Society.
• Mertz, B., Michael, L. u., Brown, M. F., & Feller, S. E. (2011). Steric and electronic influences on the torsional energy landscape of retinal. Biophysical Journal, 101(3), L17-L19.
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  PMID: 21806916;PMCID: PMC3145276;Abstract: We have performed quantum mechanical calculations for retinal model compounds to establish the rotational energy barriers for the C5-, C9-, and C13-methyl groups known to play an essential role in rhodopsin activation. Intraretinal steric interactions as well as electronic effects lower the rotational barriers of both the C9- and C13-methyl groups, consistent with experimental 2H NMR data. Each retinal methyl group has a unique rotational behavior which must be treated individually. These results are highly relevant for the parameterization of molecular mechanics force fields which form the basis of molecular dynamics simulations of retinal proteins such as rhodopsin. © 2011 Biophysical Society.
• Strtus, A. V., & Brown, M. F. (2011). Retinal Structural Dynamics in Rhodopsin Activation. Vestnik St. Petersburg University, 4(4), 229-233.
• Bartels, T., Ahlstrom, L. S., Leftin, A., Kamp, F., Haass, C., Brown, M. F., & Beyer, K. (2010). The N-terminus of the intrinsically disordered protein α-synuclein triggers membrane binding and helix folding. Biophysical Journal, 99(7), 2116-2124.
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  PMID: 20923645;PMCID: PMC3042581;Abstract: Alpha-synuclein (aS) is a 140-amino-acid protein that is involved in a number of neurodegenerative diseases. In Parkinson's disease, the protein is typically encountered in intracellular, high-molecular-weight aggregates. Although αSis abundant in the presynaptic terminals of the central nervous system, its physiological function is still unknown. There is strong evidence for the membrane affinity of the protein. One hypothesis is that lipid-induced binding and helix folding may modulate the fusion of synaptic vesicles with the presynaptic membrane and the ensuing transmitter release. Here we show that membrane recognition of the N-terminus is essential for the cooperative formation of helical domains in the protein. We used circular dichroism spectroscopy and isothermal titration calorimetry to investigate synthetic peptide fragments from different domains of the full-length aS protein. Site-specific truncation and partial cleavage of the full-length protein were employed to further characterize the structural motifs responsible for helix formation and lipid-protein interaction. Unilamellar vesicles of varying net charge and lipid compositions undergoing lateral phase separation or chain melting phase transitions in the vicinity of physiological temperatures served as model membranes. The results suggest that the membrane-induced helical folding of the first 25 residues may be driven simultaneously by electrostatic attraction and by a change in lipid ordering. Our findings highlight the significance of the aS N-terminus for folding nucleation, and provide a framework for elucidating the role of lipid-induced conformational transitions of the protein within its intracellular milieu. © 2010 by the Biophysical Society.
• Brown, M. F., Salgado, G. F., & Struts, A. V. (2010). Retinal dynamics during light activation of rhodopsin revealed by solid-state NMR spectroscopy. Biochimica et Biophysica Acta, 1798(2), 177-193.
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  PMID: 19716801;Abstract: Rhodopsin is a canonical member of class A of the G protein-coupled receptors (GPCRs) that are implicated in many of the drug interventions in humans and are of great pharmaceutical interest. The molecular mechanism of rhodopsin activation remains unknown as atomistic structural information for the active metarhodopsin II state is currently lacking. Solid-state 2H NMR constitutes a powerful approach to study atomic-level dynamics of membrane proteins. In the present application, we describe how information is obtained about interactions of the retinal cofactor with rhodopsin that change with light activation of the photoreceptor. The retinal methyl groups play an important role in rhodopsin function by directing conformational changes upon transition into the active state. Site-specific 2H labels have been introduced into the methyl groups of retinal and solid-state 2H NMR methods applied to obtain order parameters and correlation times that quantify the mobility of the cofactor in the inactive dark state, as well as the cryotrapped metarhodopsin I and metarhodopsin II states. Analysis of the angular-dependent 2H NMR line shapes for selectively deuterated methyl groups of rhodopsin in aligned membranes enables determination of the average ligand conformation within the binding pocket. The relaxation data suggest that the β-ionone ring is not expelled from its hydrophobic pocket in the transition from the pre-activated metarhodopsin I to the active metarhodopsin II state. Rather, the major structural changes of the retinal cofactor occur already at the metarhodopsin I state in the activation process. The metarhodopsin I to metarhodopsin II transition involves mainly conformational changes of the protein within the membrane lipid bilayer rather than the ligand. The dynamics of the retinylidene methyl groups upon isomerization are explained by an activation mechanism involving cooperative rearrangements of extracellular loop E2 together with transmembrane helices H5 and H6. These activating movements are triggered by steric clashes of the isomerized all-trans retinal with the β4 strand of the E2 loop and the side chains of Glu122 and Trp265 within the binding pocket. The solid-state 2H NMR data are discussed with regard to the pathway of the energy flow in the receptor activation mechanism. © 2009 Elsevier B.V. All rights reserved.
• Zaitseva, E., Brown, M. F., & Vogel, R. (2010). Sequential rearrangement of interhelical networks upon rhodopsin activation in membranes: The meta IIa conformational substate. Journal of the American Chemical Society, 132(13), 4815-4821.
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  PMID: 20230054;PMCID: PMC2859452;Abstract: Photon absorption by rhodopsin is proposed to lead to an activation pathway that is described by the extended reaction scheme Meta I → Meta II a → Meta IIb → Meta IIbH+, where Meta IIbH+ is thought to be the conformational substate that activates the G protein transducin. Here we test this extended scheme for rhodopsin in a membrane bilayer environment by investigating lipid perturbation of the activation mechanism. We found that symmetric membrane lipids having two unsaturated acyl chains, such as 1,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC), selectively stabilize the Meta IIa substate in the above mechanism. By combining FTIR and UV-visible difference spectroscopy, we characterized the structural and functional changes involved in the transition to the Meta IIa intermediate, which links the inactive Meta I intermediate with the Meta IIb states formed by helix rearrangement. Besides the opening of the Schiff base ionic lock, the Meta IIa substate is characterized by an activation switch in a conserved water-mediated hydrogen-bonded network involving transmembrane helices H1/H2/H7, which is sensed by its key residue Asp83. On the other hand, movement of retinal toward H5 and its interaction with another interhelical H3/H5 network mediated by His211 and Glu122 is absent in Meta IIa. The latter rearrangement takes place only in the subsequent transition to Meta II b, which has been previously associated with movement of H6. Our results imply that activating structural changes in the H1/H2/H7 network are triggered by disruption of the Schiff base salt bridge and occur prior to other chromophore-induced changes in the H3/H5 network and the outward tilt of H6 in the activation process. © 2010 American Chemical Society.
• Brown, M. F., Martínez-Mayorga, K., Nakanishi, K., F., G., & Struts, A. V. (2009). Retinal conformation and dynamics in activation of rhodopsin illuminated by solid-state 2H NMR spectroscopy. Photochemistry and Photobiology, 85(2), 442-453.
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  PMID: 19267870;PMCID: PMC2858981;Abstract: Solid-state NMR spectroscopy gives a powerful avenue for investigating G protein-coupled receptors and other integral membrane proteins in a native-like environment. This article reviews the use of solid-state 2H NMR to study the retinal cofactor of rhodopsin in the dark state as well as the meta I and meta II photointermediates. Site-specific 2H NMR labels have been introduced into three regions (methyl groups) of retinal that are crucially important for the photochemical function of rhodopsin. Despite its phenomenal stability 2H NMR spectroscopy indicates retinal undergoes rapid fluctuations within the protein binding cavity. The spectral lineshapes reveal the methyl groups spin rapidly about their three-fold (C3) axes with an order parameter for the off-axial motion of For the dark state, the 2H NMR structure of 11-cis-retinal manifests torsional twisting of both the polyene chain and the β-ionone ring due to steric interactions of the ligand and the protein. Retinal is accommodated within the rhodopsin binding pocket with a negative pretwist about the C11=C12 double bond. Conformational distortion explains its rapid photochemistry and reveals the trajectory of the 11-cis to trans isomerization. In addition, 2H NMR has been applied to study the retinylidene dynamics in the dark and light-activated states. Upon isomerization there are drastic changes in the mobility of all three methyl groups. The relaxation data support an activation mechanism whereby the β-ionone ring of retinal stays in nearly the same environment, without a large displacement of the ligand. Interactions of the β-ionone ring and the retinylidene Schiff base with the protein transmit the force of the retinal isomerization. Solid-state 2H NMR thus provides information about the flow of energy that triggers changes in hydrogen-bonding networks and helix movements in the activation mechanism of the photoreceptor. © 2009 The Authors.
• Brownholland, D. P., Longo, G. S., Struts, A. V., Justice, M. J., Szleifer, I., Petrache, H. I., Brown, M. F., & Thompson, D. H. (2009). Phase separation in binary mixtures of bipolar and monopolar lipid dispersions revealed by 2H NMR spectroscopy, small angle x-ray scattering, and molecular theory. Biophysical Journal, 97(10), 2700-2709.
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  PMID: 19917223;PMCID: PMC2776299;Abstract: Binary mixtures of C20BAS and POPC membranes were studied by solid-state 2H NMR spectroscopy and small angle x-ray scattering (SAXS) over a wide range of concentrations and at different temperatures. Three specifically deuterated C20BAS derivatives-[1′,1′, 20′,20′-2H4]C20BAS, [2′,2′,19′,19′-2H4]C 20BAS, and [10′,11′-2H2]C 20BAS - combined with protiated 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC), as well as membranes containing POPC-d31 and fully protiated bolalipid, were used in NMR experiments to obtain structural information for the mixtures. The 2H NMR spectra of [10′,11′-2H2]C20BAS/POPC membrane dispersions reveal that the bolalipid is predominantly in the transmembrane conformation at high bolalipid concentrations (100, 90, and 70 mol %). At ≤50 mol % C20BAS, smaller quadrupolar couplings appear in the spectra, indicating the presence of U-shaped conformers. The proportion of U-shaped bolalipids increases as the amount of POPC in the membrane increases; however, the transmembrane component remains the dominant bolalipid conformation in the membrane even at 45°C and 10 mol % C20BAS, where it accounts for ∼50% of the bolalipid population. The large fraction of C20BAS transmembrane conformers, regardless of the C20BAS/POPC ratio, together with the findings from molecular mean-field theory calculations, suggests the coexistence of phase-separated bolalipid-rich domains and POPC-rich domains. A single lamellar repeat distance was observed in SAXS experiments corresponding to the average repeat spacing expected for C20BAS-and POPC-rich domains. These observations are consistent with the presence of microphase-separated domains in the mixed membrane samples that arise from POPC-C20BAS hydrophobic mismatch. © 2009 by the Biophysical Society.
• Bartels, T., Lankalapalli, R. S., Bittman, R., Beyer, K., & Brown, M. F. (2008). Raftlike mixtures of sphingomyelin and cholesterol investigated by solid-state 2H NMR spectroscopy. Journal of the American Chemical Society, 130(44), 14521-14532.
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  PMID: 18839945;PMCID: PMC2756786;Abstract: Sphingomyelin is a lipid that is abundant in the nervous systems of mammals, where it is associated with putative microdomains in cellular membranes and undergoes alterations due to aging or neurodegeneration. We investigated the effect of varying the concentration of cholesterol in binary and ternary mixtures with N-palmitoylsphingomyelin (PSM) and 1-palmitoyl-2-oleoyl-sn- glycero-3-phosphocholine (POPC) using deuterium nuclear magnetic resonance (2H NMR) spectroscopy in both macroscopically aligned and unoriented multilamellar dispersions. In our experiments, we used PSM and POPC perdeuterated on the N-acyl and sn-1 acyl chains, respectively. By measuring solid-state 2H NMR spectra of the two lipids separately in mixtures with the same compositions as a function of cholesterol mole fraction and temperature, we obtained clear evidence for the coexistence of two liquid-crystalline domains in distinct regions of the phase diagram. According to our analysis of the first moments M1 and the observed 2H NMR spectra, one of the domains appears to be a liquid-ordered phase. We applied a mean-torque potential model as an additional tool to calculate the average hydrocarbon thickness, the area per lipid, and structural parameters such as chain extension and thermal expansion coefficient in order to further define the two coexisting phases. Our data imply that phase separation takes place in raftlike ternary PSM/POPC/cholesterol mixtures over a broad temperature range but vanishes at cholesterol concentrations equal to or greater than a mole fraction of 0.33. Cholesterol interacts preferentially with sphingomyelin only at smaller mole fractions, above which a homogeneous liquid-ordered phase is present. The reasons for these phase separation phenomena seem to be differences in the effects of cholesterol on the configurational order of the palmitoyl chains in PSM-d31 and POPC-d31 and a difference in the affinity of cholesterol for sphingomyelin observed at low temperatures. Hydrophobic matching explains the occurrence of raftlike domains in cellular membranes at intermediate cholesterol concentrations but not saturating amounts of cholesterol. © 2008 American Chemical Society.
• Holland, D. P., Struts, A. V., Brown, M. F., & Thompson, D. H. (2008). Bolalipid membrane structure revealed by solid-state 2H NMR spectroscopy. Journal of the American Chemical Society, 130(14), 4584-4585.
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  PMID: 18348566;Abstract: Membranes made from three specifically deuterium-labeled ether-linked bolalipids, [1′,1′,20′,20′-2H4]C20BAS-PC, [2′,2′,19′,19′-2H4]C20BAS-PC, or [10′,11′-2H2]C20BAS-PC, were analyzed by 2H NMR spectroscopy. Unlike more common monopolar, ester-linked phospholipids, C20BAS-PC exhibits a high degree of orientational order throughout the membrane and the sn-1 chain of the lipid initially penetrates the bilayer at an orientation different from that of the bilayer normal, resulting in inequivalent deuterium atoms at the C1 position. The approximate hydrophobic layer thickness and area per lipid are 18.4 Å and 60.4 Å2, respectively, at 25 °C, and their respective thermal expansion coefficients are within 20% of the monopolar phospholipid, DLPC. Copyright © 2008 American Chemical Society.
• Kobayashi, M., Struts, A. V., Fujiwara, T., Brown, M. F., & Akutsu, H. (2008). Fluid mechanical matching of H+-ATP synthase subunit c-ring with lipid membranes revealed by 2H solid-state NMR. Biophysical Journal, 94(11), 4339-4347.
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  PMID: 18310246;PMCID: PMC2480690;Abstract: The F 1 F 0-ATP synthase utilizes the transmembrane H + gradient for the synthesis of ATP. F 0 subunit c-ring plays a key role in transporting H + through F 0 in the membrane. We investigated the interactions of Escherichia coli subunit c with dimyristoylphosphatidylcholine (DMPC-d 54) at lipid/protein ratios of 50:1 and 20:1 by means of 2H-solid-state NMR. In the liquid-crystalline state of DMPC, the 2H-NMR moment values and the order parameter (S CD) profile were little affected by the presence of subunit c, suggesting that the bilayer thickness in the liquid-crystalline state is matched to the transmembrane hydrophobic surface of subunit c. On the other hand, hydrophobic mismatch of subunit c with the lipid bilayer was observed in the gel state of DMPC. Moreover, the viscoelasticity represented by a square-law function of the 2H-NMR relaxation was also little influenced by subunit c in the fluid phase, in contrast with flexible nonionic detergents or rigid additives. Thus, the hydrophobic matching of the lipid bilayer to subunit c involves at least two factors, the hydrophobic length and the fluid mechanical property. These findings may be important for the torque generation in the rotary catalytic mechanism of the F 1F 0-ATPse molecular motor. © 2008 by the Biophysical Society.
• Mahalingam, M., Martínez-Mayorga, K., Brown, M. F., & Vogel, R. (2008). Two protonation switches control rhodopsin activation in membranes. Proceedings of the National Academy of Sciences of the United States of America, 105(46), 17795-17800.
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  PMID: 18997017;PMCID: PMC2584695;Abstract: Activation of the G protein-coupled receptor (GPCR) rhodopsin is initiated by light-induced isomerization of the retinal ligand, which triggers 2 protonation switches in the conformational transition to the active receptor state Meta II. The first switch involves disruption of an interhelical salt bridge by internal proton transfer from the retinal protonated Schiff base (PSB) to its counterion, Glu-113, in the transmembrane domain. The second switch consists of uptake of a proton from the solvent by Glu-134 of the conserved E(D)RY motif at the cytoplasmic terminus of helix 3, leading to pH-dependent receptor activation. By using a combination of UV-visible and FTIR spectroscopy, we study the activation mechanism of rhodopsin in different membrane environments and show that these 2 protonation switches become partially uncoupled at physiological temperature. This partial uncoupling leads to ≈50% population of an entropy-stabilized Meta II state in which the interhelical PSB salt bridge is broken and activating helix movements have taken place but in which Glu-134 remains unprotonated. This partial activation is converted to full activation only by coupling to the pH-dependent protonation of Glu-134 from the solvent, which stabilizes the active receptor conformation by lowering its enthalpy. In a membrane environment, protonation of Glu-134 is therefore a thermodynamic rather than a structural prerequisite for activating helix movements. In light of the conservation of the E(D)RY motif in rhodopsin-like GPCRs, protonation of this carboxylate also may serve a similar function in signal transduction of other members of this receptor family. © 2008 by The National Academy of Sciences of the USA.
• Michel, R., Subramaniam, V., McArthur, S. L., Bondurant, B., D'Ambruoso, G. D., Hall Jr., H. K., Brown, M. F., Ross, E. E., Saavedra, S. S., & Castner, D. G. (2008). Ultra-high vacuum surface analysis study of rhodopsin incorporation into supported lipid bilayers. Langmuir, 24(9), 4901-4906.
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  PMID: 18393486;PMCID: PMC2722912;Abstract: Planar supported lipid bilayers that are stable under ambient atmospheric and ultra-high-vacuum conditions were prepared by cross-linking polymerization of bis-sorbylphosphatidylcholine (bis-SorbPC). X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS) were employed to investigate bilayers that were cross-linked using either redox-initiated radical polymerization or ultraviolet photopolymerization. The redox method yields a more structurally intact bilayer; however, the UV method is more compatible with incorporation of transmembrane proteins. UV polymerization was therefore used to prepare cross-linked bilayers with incorporated bovine rhodopsin, a light-activated, G-protein-coupled receptor (GPCR). A previous study (Subramaniam, V.; Alves, I. D.; Salgado, G. F. J.; Lau, P. W.; Wysocki, R. J.; Salamon, Z.; Tollin, G.; Hruby, V. J.; Brown, M. F.; Saavedra, S. S. J. Am. Chem. Soc. 2005, 127, 5320-5321) showed that rhodopsin retains photoactivity after incorporation into UV-polymerized bis-SorbPC, but did not address how the protein is associated with the bilayer. In this study, we show that rhodopsin is retained in supported bilayers of poly(bis-SorbPC) under ultra-high-vacuum conditions, on the basis of the increase in the XPS nitrogen concentration and the presence of characteristic amino acid peaks in the ToF-SIMS data. Angle-resolved XPS data show that the protein is inserted into the bilayer, rather than adsorbed on the bilayer surface. This is the first study to demonstrate the use of ultra-high-vacuum techniques for structural studies of supported proteolipid bilayers. © 2008 American Chemical Society.
• Subramaniam, V., D'Ambruoso, G. D., Hall Jr., H. K., Wysocki Jr., R. J., Brown, M. F., & Saavedra, S. S. (2008). Reconstitution of rhodopsin into polymerizable planar supported lipid bilayers: Influence of dienoyl monomer structure on photoactivation. Langmuir, 24(19), 11067-11075.
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  PMID: 18759470;PMCID: PMC2726791;Abstract: G-protein-coupled receptors (GPCRs) play key roles in cellular signal transduction and many are pharmacologically important targets for drug discovery. GPCRs can be reconstituted in planar supported lipid bilayers (PSLBs) with retention of activity, which has led to development of GPCR-based biosensors and biochips. However, PSLBs composed of natural lipids lack the high stability desired for many technological applications. One strategy is to use synthetic lipid monomers that can be polymerized to form robust bilayers. A key question is how lipid polymerization affects GPCR structure and activity. Here we have investigated the photochemical activity of bovine rhodopsin (Rhô), a model GPCR, reconstituted into PSLBs composed of lipids having one or two polymerizable dienoyl moieties located in different regions of the acyl chains. Plasmon waveguide resonance spectroscopy was used to compare the degree of Rho photoactivation in fluid and poly(lipid) PSLBs. The position of the dienoyl moiety was found to have a significant effect: polymerization near the glycerol backbone significantly attenuates Rho activity whereas polymerization near the acyl chain termini does not. Differences in cross-link density near the acyl chain termini also do not affect Rho activity. In unpolymerized PSLBs, an equimolar mixture of phosphatidylethanolamine and phosphatidylcholine (PC) lipids enhances activity relative to pure PC; however after polymerization, the enhancement is eliminated which is attributed to stabilization of the membrane lamellar phase. These results should provide guidance for the design of robust lipid bilayers functionalized with transmembrane proteins for use in membrane-based biochips and biosensors. © 2008 American Chemical Society.
• Brown, M. F., Brown, M., Struts, A. V., Salgado, G. F., Tanaka, K., Krane, S., & Nakanishi, K. (2007). Structural analysis and dynamics of retinal chromophore in dark and meta I states of rhodopsin from 2H NMR of aligned membranes. Journal of Molecular Biology, 372(1).
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  Rhodopsin is a prototype for G protein-coupled receptors (GPCRs) that are implicated in many biological responses in humans. A site-directed (2)H NMR approach was used for structural analysis of retinal within its binding cavity in the dark and pre-activated meta I states. Retinal was labeled with (2)H at the C5, C9, or C13 methyl groups by total synthesis, and was used to regenerate the opsin apoprotein. Solid-state (2)H NMR spectra were acquired for aligned membranes in the low-temperature lipid gel phase versus the tilt angle to the magnetic field. Data reduction assumed a static uniaxial distribution, and gave the retinylidene methyl bond orientations plus the alignment disorder (mosaic spread). The dark-state (2)H NMR structure of 11-cis-retinal shows torsional twisting of the polyene chain and the beta-ionone ring. The ligand undergoes restricted motion, as evinced by order parameters of approximately 0.9 for the spinning C-C(2)H(3) groups, with off-axial fluctuations of approximately 15 degrees . Retinal is accommodated within the rhodopsin binding pocket with a negative pre-twist about the C11=C12 double bond that explains its rapid photochemistry and the trajectory of 11-cis to trans isomerization. In the cryo-trapped meta I state, the (2)H NMR structure shows a reduction of the polyene strain, while torsional twisting of the beta-ionone ring is maintained. Distortion of the retinal conformation is interpreted through substituent control of receptor activation. Steric hindrance between trans retinal and Trp265 can trigger formation of the subsequent activated meta II state. Our results are pertinent to quantum and molecular mechanics simulations of ligands bound to GPCRs, and illustrate how (2)H NMR can be applied to study their biological mechanisms of action.
• Brown, M. F., Heyn, M. P., Job, C., Kim, S., Moltke, S., Nakanishi, K., Nevzorov, A. A., Struts, A. V., Salgado, G. F., & Wallat, I. (2007). Solid-State 2H NMR spectroscopy of retinal proteins in aligned membranes. Biochimica et Biophysica Acta, 1768(12), 2979-3000.
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  PMID: 18021739;Abstract: Solid-state 2H NMR spectroscopy gives a powerful avenue to investigating the structures of ligands and cofactors bound to integral membrane proteins. For bacteriorhodopsin (bR) and rhodopsin, retinal was site-specifically labeled by deuteration of the methyl groups followed by regeneration of the apoprotein. 2H NMR studies of aligned membrane samples were conducted under conditions where rotational and translational diffusion of the protein were absent on the NMR time scale. The theoretical lineshape treatment involved a static axial distribution of rotating C-C2H3 groups about the local membrane frame, together with the static axial distribution of the local normal relative to the average normal. Simulation of solid-state 2H NMR lineshapes gave both the methyl group orientations and the alignment disorder (mosaic spread) of the membrane stack. The methyl bond orientations provided the angular restraints for structural analysis. In the case of bR the retinal chromophore is nearly planar in the dark- and all-trans light-adapted states, as well upon isomerization to 13-cis in the M state. The C13-methyl group at the "business end" of the chromophore changes its orientation to the membrane upon photon absorption, moving towards W182 and thus driving the proton pump in energy conservation. Moreover, rhodopsin was studied as a prototype for G protein-coupled receptors (GPCRs) implicated in many biological responses in humans. In contrast to bR, the retinal chromophore of rhodopsin has an 11-cis conformation and is highly twisted in the dark state. Three sites of interaction affect the torsional deformation of retinal, viz. the protonated Schiff base with its carboxylate counterion; the C9-methyl group of the polyene; and the β-ionone ring within its hydrophobic pocket. For rhodopsin, the strain energy and dynamics of retinal as established by 2H NMR are implicated in substituent control of activation. Retinal is locked in a conformation that is twisted in the direction of the photoisomerization, which explains the dark stability of rhodopsin and allows for ultra-fast isomerization upon absorption of a photon. Torsional strain is relaxed in the meta I state that precedes subsequent receptor activation. Comparison of the two retinal proteins using solid-state 2H NMR is thus illuminating in terms of their different biological functions. © 2007 Elsevier B.V. All rights reserved.
• Brown, M., Brown, M. F., Lau, P., Grossfield, A., Feller, S. E., & Pitman, M. C. (2007). Dynamic structure of retinylidene ligand of rhodopsin probed by molecular simulations. Journal of Molecular Biology, 372(4).
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  Rhodopsin is currently the only available atomic-resolution template for understanding biological functions of the G protein-coupled receptor (GPCR) family. The structural basis for the phenomenal dark state stability of 11-cis-retinal bound to rhodopsin and its ultrafast photoreaction are active topics of research. In particular, the beta-ionone ring of the retinylidene inverse agonist is crucial for the activation mechanism. We analyzed a total of 23 independent, 100 ns all-atom molecular dynamics simulations of rhodopsin embedded in a lipid bilayer in the microcanonical (N,V,E) ensemble. Analysis of intramolecular fluctuations predicts hydrogen-out-of-plane (HOOP) wagging modes of retinal consistent with those found in Raman vibrational spectroscopy. We show that sampling and ergodicity of the ensemble of simulations are crucial for determining the distribution of conformers of retinal bound to rhodopsin. The polyene chain is rigidly locked into a single, twisted conformation, consistent with the function of retinal as an inverse agonist in the dark state. Most surprisingly, the beta-ionone ring is mobile within its binding pocket; interactions are non-specific and the cavity is sufficiently large to enable structural heterogeneity. We find that retinal occupies two distinct conformations in the dark state, contrary to most previous assumptions. The beta-ionone ring can rotate relative to the polyene chain, thereby populating both positively and negatively twisted 6-s-cis enantiomers. This result, while unexpected, strongly agrees with experimental solid-state (2)H NMR spectra. Correlation analysis identifies the residues most critical to controlling mobility of retinal; we find that Trp265 moves away from the ionone ring prior to any conformational transition. Our findings reinforce how molecular dynamics simulations can challenge conventional assumptions for interpreting experimental data, especially where existing models neglect conformational fluctuations.
• Petrache, H. I., & Brown, M. F. (2007). X-ray scattering and solid-state deuterium nuclear magnetic resonance probes of structural fluctuations in lipid membranes. Methods in Molecular Biology, 400, 341-353.
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  PMID: 17951745;Abstract: Molecular fluctuations are a dominant feature of biomembranes. Cellular functions might rely on these properties in ways yet to be determined. This expectation is suggested by the fact that membrane deformation and rigidity, which govern molecular fluctuations, have been implicated in a number of cellular functions. However, fluctuations are more challenging to measure than average structures, which partially explain the small number of dedicated studies. Here, it is shown that two accessible laboratory methods, small-angle X-ray scattering and solid-state deuterium nuclear magnetic resonance (NMR), can be used as complementary probes of structural fluctuations in lipid membranes. In the case of X-ray scattering, membrane undulations give rise to logarithmically varying positional correlations that generate scattering peaks with long (power-law) tails. In the case of 2H NMR spectroscopy, fluctuations in the magnetic-coupling energies resulting from molecular motions cause relaxation among the various spin energy levels, and yield a powerful probe of orientational fluctuations of the lipid molecules. A unified interpretation of the combined scattering and 2H NMR data is provided by a liquid-crystalline membrane deformation model. The importance of this approach is that it is possible to utilize a microscopic model for positional and orientational observables to calculate bulk material properties of liquid-crystalline systems. © Humana Press Inc.
• Tanaka, K., Struts, A. V., Krane, S., Fujioka, N., F., G., Martínez-Mayorga, K., Brown, M. F., & Nakanishi, K. (2007). Synthesis of CD3-labeled 11-cis-retinals and application to solid-state deuterium NMR spectroscopy of rhodopsin. Bulletin of the Chemical Society of Japan, 80(11), 2177-2184.
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  Abstract: Efficient synthesis of 11-Z-retinals labeled with 2H at the C5, C9, or C13 methyl groups is described. The 2H-labeled retinals were used to regenerate the visual pigment rhodopsin for structural investigations. Solid-state 2H NMR data provided the orientation of retinal within the rhodopsin binding pocket as well as its conformation. Extension of the approach to other membrane receptors can yield knowledge of their mechanisms of activation as a guide for ligand-based drug design. © 2007 The Chemical Society of Japan.
• Vogel, A., Tan, K., Waldmann, H., Feller, S. E., Brown, M. F., & Huster, D. (2007). Flexibility of ras lipid modifications studied by 2H solid-state NMR and molecular dynamics simulations. Biophysical Journal, 93(8), 2697-2712.
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  PMID: 17557790;PMCID: PMC1989704;Abstract: Human posttranslationally modified N-ras oncogenes are known to be implicated in numerous human cancers. Here, we applied a combination of experimental and computational techniques to determine structural and dynamical details of the lipid chain modifications of an N-ras heptapeptide in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) membranes. Experimentally, 2H NMR spectroscopy was used to study oriented membranes that incorporated ras heptapeptides with two covalently attached perdeuterated hexadecyl chains. Atomistic molecular dynamics simulations of the same system were carried out over 100 ns including 60 DMPC and 4 ras molecules. Several structural and dynamical experimental parameters could be directly compared to the simulation. Experimental and simulated 2H NMR order parameters for the methylene groups of the ras lipid chains exhibited a systematic difference attributable to the absence of collective motions in the simulation and to geometrical effects. In contrast, experimental 2H NMR spin-lattice relaxation rates for Zeeman order were well reproduced in the simulation. The lack of slower collective motions in the simulation did not appreciably influence the relaxation rates at a Larmor frequency of 115.1 MHz. The experimental angular dependence of the 2H NM Rrelaxation rates with respect to the external magnetic field was also relatively well simulated. These relaxation rates showed a weak angular dependence, suggesting that the lipid modifications of ras are very flexible and highly mobile in agreement with the low order parameters. To quantify these results, the angular dependence of the 2H relaxation rates was calculated by an analytical model considering both molecular and collective motions. Peptide dynamics in the membrane could be modeled by an anisotropic diffusion tensor with principal values of D∥ = 2.1 × 109 s-1 and D⊥ = 4.5 × 105 s-1. A viscoelastic fitting parameter describing the membrane elasticity, viscosity, and temperature was found to be relatively similar for the ras peptide and the DMPC host matrix. Large motional amplitudes and relatively short correlation times facilitate mixing and dispersal with the lipid bilayer matrix, with implications for the role of the full-length ras protein in signal transduction and oncogenesis. © 2007 by the Biophysical Society.
• Brown, M., Botelho, A. V., Huber, T., Sakmar, T. P., & Brown, M. F. (2006). Curvature and hydrophobic forces drive oligomerization and modulate activity of rhodopsin in membranes. Biophysical journal, 91(12).
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  G protein-coupled receptors (GPCRs) are essential components of cellular signaling pathways. They are the targets of many current pharmaceuticals and are postulated to dimerize or oligomerize in cellular membranes in conjunction with their functional mechanisms. We demonstrate using fluorescence resonance energy transfer how association of rhodopsin occurs by long-range lipid-protein interactions due to geometrical forces, yielding greater receptor crowding. Constitutive association of rhodopsin is promoted by a reduction in membrane thickness (hydrophobic mismatch), but also by an increase in protein/lipid molar ratio, showing the importance of interactions extending well beyond a single annulus of boundary lipids. The fluorescence data correlate with the pK(a) for the MI-to-MII transition of rhodopsin, where deprotonation of the retinylidene Schiff base occurs in conjunction with helical movements leading to activation of the photoreceptor. A more dispersed membrane environment optimizes formation of the MII conformation that results in visual function. A flexible surface model explains both the dispersal and activation of rhodopsin in terms of bilayer curvature deformation (strain) and hydrophobic solvation energy. The bilayer stress is related to the lateral pressure profile in terms of the spontaneous curvature and associated bending rigidity. Transduction of the strain energy (frustration) of the bilayer drives protein oligomerization and conformational changes in a coupled manner. Our findings illuminate the physical principles of membrane protein association due to chemically nonspecific interactions in fluid lipid bilayers. Moreover, they yield a conceptual framework for understanding how the tightly regulated lipid compositions of cellular membranes influence their protein-mediated functions.
• Brown, M., Martínez-Mayorga, K., Pitman, M. C., Grossfield, A., Feller, S. E., & Brown, M. F. (2006). Retinal counterion switch mechanism in vision evaluated by molecular simulations. Journal of the American Chemical Society, 128(51).
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  Photoisomerization of the retinylidene chromophore of rhodopsin is the starting point in the vision cascade. A counterion switch mechanism that stabilizes the retinal protonated Schiff base (PSB) has been proposed to be an essential step in rhodopsin activation. On the basis of vibrational and UV-visible spectroscopy, two counterion switch models have emerged. In the first model, the PSB is stabilized by Glu181 in the meta I state, while in the most recent proposal, it is stabilized by Glu113 as well as Glu181. We assess these models by conducting a pair of microsecond scale, all-atom molecular dynamics simulations of rhodopsin embedded in a 99-lipid bilayer of SDPC, SDPE, and cholesterol (2:2:1 ratio) varying the starting protonation state of Glu181. Theoretical simulations gave different orientations of retinal for the two counterion switch mechanisms, which were used to simulate experimental 2H NMR spectra for the C5, C9, and C13 methyl groups. Comparison of the simulated 2H NMR spectra with experimental data supports the complex-counterion mechanism. Hence, our results indicate that Glu113 and Glu181 stabilize the retinal PSB in the meta I state prior to activation of rhodopsin.
• Brown, M., Salgado, G. F., Struts, A. V., Tanaka, K., Krane, S., Nakanishi, K., & Brown, M. F. (2006). Solid-state 2H NMR structure of retinal in metarhodopsin I. Journal of the American Chemical Society, 128(34).
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  The structural and photochemical changes in rhodopsin due to absorption of light are crucial for understanding the process of visual signaling. We investigated the structure of trans-retinal in the metarhodopsin I photointermediate (MI), where the retinylidene cofactor functions as an antagonist. Rhodopsin was regenerated using retinal that was (2)H-labeled at the C5, C9, or C13 methyl groups and was reconstituted with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine. Membranes were aligned by isopotential centrifugation, and rhodopsin in the supported bilayers was then bleached and cryotrapped in the MI state. Solid-state (2)H NMR spectra of oriented rhodopsin in the low-temperature lipid gel state were analyzed in terms of a static uniaxial distribution (Nevzorov, A. A.; Moltke, S.; Heyn, M. P.; Brown, M. F. J. Am. Chem. Soc. 1999, 121, 7636-7643). The line shape analysis allowed us to obtain the methyl bond orientations relative to the membrane normal in the presence of substantial alignment disorder (mosaic spread). Relative orientations of the methyl groups were used to calculate effective torsional angles between the three different planes that represent the polyene chain and the beta-ionone ring of retinal. Assuming a three-plane model, a less distorted structure was found for retinal in MI compared to the dark state. Our results are pertinent to how photonic energy is channeled within the protein to allow the strained retinal conformation to relax, thereby forming the activated state of the receptor.
• Martínez-Mayorga, K., Pitman, M. C., Grossfield, A., Feller, S. E., & Brown, M. F. (2006). Retinal counterion switch mechanism in vision evaluated by molecular simulations. Journal of the American Chemical Society, 128(51), 16502-16503.
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  PMID: 17177390;Abstract: Photoisomerization of the retinylidene chromophore of rhodopsin is the starting point in the vision cascade. A counterion switch mechanism that stabilizes the retinal protonated Schiff base (PSB) has been proposed to be an essential step in rhodopsin activation. On the basis of vibrational and UV-visible spectroscopy, two counterion switch models have emerged. In the first model, the PSB is stabilized by Glu181 in the meta I state, while in the most recent proposal, it is stabilized by Glu113 as well as Glu181. We assess these models by conducting a pair of microsecond scale, all-atom molecular dynamics simulations of rhodopsin embedded in a 99-lipid bilayer of SDPC, SDPE, and cholesterol (2:2:1 ratio) varying the starting protonation state of Glu181. Theoretical simulations gave different orientations of retinal for the two counterion switch mechanisms, which were used to simulate experimental 2H NMR spectra for the C5, C9, and C13 methyl groups. Comparison of the simulated 2H NMR spectra with experimental data supports the complex-counterion mechanism. Hence, our results indicate that Glu113 and Glu181 stabilize the retinal PSB in the meta I state prior to activation of rhodopsin. Copyright © 2006 American Chemical Society.
• Alves, I. D., Salgado, G. F., Salamon, Z., Brown, M. F., Tollin, G., & Hruby, V. J. (2005). Phosphatidylethanolamine Enhances Rhodopsin Photoactivation and Transducin Binding in a Solid-Supported Lipid Bilayer as Determined Using Plasmon-Waveguide Resonance Spectroscopy. Biophysical Journal, 88, 198–210.
• Brown, M., Brown, M. F., Rajamoorthi, K., Petrache, H. I., & McIntosh, T. J. (2005). Packing and viscoelasticity of polyunsaturated omega-3 and omega-6 lipid bilayers as seen by 2H NMR and X-ray diffraction. Journal of the American Chemical Society, 127(5).
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  Polyunsaturated phospholipids of the omega-3 and omega-6 classes play key roles in cellular functions, yet their mechanisms of biological action are still a matter of debate. Using deuterium ((2)H) NMR spectroscopy and small-angle X-ray diffraction, we show how membrane properties are modified by docosahexaenoic (DHA; 22:6) and arachidonic (AA; 20:4) acyl chains of the omega-3 and the omega-6 families, respectively. Structural and dynamical differences due to polyunsaturation are evident in both the ordered and disordered phases of mixed-chain (16:0)(22:6)PC and (16:0)(20:4)PC bilayers. Due to the lower chain melting temperature, the omega-6 AA bilayer is more disordered in the fluid (L(alpha)) state than the omega-3 DHA bilayer; it is thinner with a larger area per lipid. The thermal hysteresis observed for the DHA bilayer may represent the influences of angle-iron conformers in the gel state and back-bended, hairpinlike conformers in the fluid state, consistent with molecular dynamics studies. Interpretation of the (2)H NMR order profiles of (16:0-d(31))(22:6)PC and (16:0-d(31))(20:4)PC together with X-ray electron density profiles reveals an uneven distribution of mass; i.e., the sn-1 saturated chain is displaced toward the membrane center, whereas the sn-2 polyunsaturated chain is shifted toward the bilayer aqueous interface. Moreover, the (2)H NMR relaxation rates are increased by the presence of omega-6 AA chains compared to omega-3 DHA chains. When evaluated at the same amplitude of motion, relaxation parameters give a naturally calibrated scale for comparison of fluid lipid bilayers. Within this framework, polyunsaturated bilayers are relatively soft to bending and area fluctuations on the mesoscale approaching molecular dimensions. Significant differences are evident in the viscoelastic properties of the omega-3 and omega-6 bilayers, a possibly biologically relevant feature that distinguishes between the two phospholipid classes.
• Subramaniam, V., Alves, I. D., Salgado, G. F., Lau, P. W., Wysocki, Jr, R. J., Salamon, Z., Tollin, G., Hruby, V. J., Brown, M. F., & Saavedra, S. S. (2005). Rhodopsin Reconstituted into a Planar-Supported Lipid Bilayer Retains Photoactivity after Cross-Linking Polymerization of Lipid Monomers. Journal of the American Chemical Society, 127, 5320-5321.
• Vogel, A., Katzka, C. P., Waldmann, H., Arnold, K., Brown, M. F., & Huster, D. (2005). Lipid modifications of a ras peptide exhibit altered packing and mobility versus host membrane as detected by 2H solid-state NMR. Journal of the American Chemical Society, 127(35), 12263-12272.
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  PMID: 16131204;Abstract: The human N-ras protein binds to cellular membranes by insertion of two covalently bound posttranslational lipid modifications, which is crucial for its function in signal transduction and cell proliferation. Mutations in ras may lead to unregulated cell growth and eventually cancer, making it an important therapeutic target. Here we have investigated the molecular details of the membrane binding mechanism. A heptapeptide derived from the C-terminus of the human N-ras protein was synthesized including two hexadecyl modifications. Solid-state 2H NMR was used to determine the packing and molecular dynamics of the ras lipid chains as well as the phospholipid matrix. Separately labeling the chains of the peptide and the phospholipids with 2H enabled us to obtain atomically resolved parameters relevant to their structural dynamics. While the presence of ras only marginally affected the packing of DMPC membranes, dramatically lower order parameters (SCD) were observed for the ras acyl chains indicating modified packing properties. Essentially identical projected lengths of the 16:0 ras chains and the 14:0 DMPC chains were found, implying that the polypeptide backbone is located at the lipid-water interface. Dynamical properties of both the ras and phospholipid chains were determined from spin-lattice 2H relaxation (R 1Z) measurements. Plots of R1Z rates versus the corresponding squared segmental order parameters revealed striking differences. We propose the ras peptide is confined to microdomains containing DMPC chains which are in exchange with the bulk bilayer on the 2H NMR time scale (∼10-5 s). Compared to the host DMPC matrix, the ras lipid modifications are extremely flexible and undergo relatively large amplitude motions. It is hypothesized that this flexibility is a requirement for the optimal anchoring of lipid-modified proteins to cellular membranes. © 2005 American Chemical Society.
• Brown, M., Salgado, G. F., Struts, A. V., Tanaka, K., Fujioka, N., Nakanishi, K., & Brown, M. F. (2004). Deuterium NMR structure of retinal in the ground state of rhodopsin. Biochemistry, 43(40).
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  The conformation of retinal bound to the G protein-coupled receptor rhodopsin is intimately linked to its photochemistry, which initiates the visual process. Site-directed deuterium ((2)H) NMR spectroscopy was used to investigate the structure of retinal within the binding pocket of bovine rhodopsin. Aligned recombinant membranes were studied containing rhodopsin that was regenerated with retinal (2)H-labeled at the C(5), C(9), or C(13) methyl groups by total synthesis. Studies were conducted at temperatures below the gel to liquid-crystalline phase transition of the membrane lipid bilayer, where rotational and translational diffusion of rhodopsin is effectively quenched. The experimental tilt series of (2)H NMR spectra were fit to a theoretical line shape analysis [Nevzorov, A. A., Moltke, S., Heyn, M. P., and Brown, M. F. (1999) J. Am. Chem. Soc. 121, 7636-7643] giving the retinylidene bond orientations with respect to the membrane normal in the dark state. Moreover, the relative orientations of pairs of methyl groups were used to calculate effective torsional angles between different planes of unsaturation of the retinal chromophore. Our results are consistent with significant conformational distortion of retinal, and they have important implications for quantum mechanical calculations of its electronic spectral properties. In particular, we find that the beta-ionone ring has a twisted 6-s-cis conformation, whereas the polyene chain is twisted 12-s-trans. The conformational strain of retinal as revealed by solid-state (2)H NMR is significant for explaining the quantum yields and mechanism of its ultrafast photoisomerization in visual pigments. This work provides a consensus view of the retinal conformation in rhodopsin as seen by X-ray diffraction, solid-state NMR spectroscopy, and quantum chemical calculations.
• Henzler-Wildman, K. A., Martinez, G. V., Brown, M. F., & Ramamoorthy, A. (2004). Perturbation of the hydrophobic core of lipid bilayers by the human antimicrobial peptide LL-37. Biochemistry, 43(26), 8459-8469.
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  PMID: 15222757;Abstract: LL-37 is a cationic, amphipathic α-helical antimicrobial peptide found in humans that kills cells by disrupting the cell membrane. To disrupt membranes, antimicrobial peptides such as LL-37 must alter the hydrophobic core of the bilayer. Differential scanning calorimetry and deuterium (2H) NMR experiments on acyl chain perdeuterated lipids demonstrate that LL-37 inserts into the hydrophobic region of the bilayer and alters the chain packing and cooperativity. The results show that hydrophobic interactions between LL-37 and the hydrophobic acyl chains are as important for the ability of this peptide to disrupt lipid bilayers as its electrostatic interactions with the polar headgroups. The 2H NMR data are consistent with the previously determined surface orientation of LL-37 (Henzler Wildman, K. A., et al. (2003) Biochemistry 42, 6545) with an estimated 5-6 Å depth of penetration of the hydrophobic face of the amphipathic helix into the hydrophobic interior of the bilayer. LL-37 also alters the material properties of lipid bilayers, including the area per lipid, hydrophobic thickness, and coefficient of thermal expansion in a manner that varies with lipid type and temperature. Comparison of the effect of LL-37 on 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC-d 31) and 1,2-dimyristoyl-phosphatidylcholine (DMPC-d54) at different temperatures demonstrates the importance of bilayer order in determining the type and extent of disordering and disruption of the hydrophobic core by LL-37. One possible explanation, which accounts for both the 2H NMR data presented here and the known surface orientation of LL-37 under identical conditions, is that bilayer order influences the depth of insertion of LL-37 into the hydrophobic/hydrophilic interface of the bilayer, altering the balance of electrostatic and hydrophobic interactions between the peptide and the lipids.
• Huber, T., Botelho, A. V., Beyer, K., & Brown, M. F. (2004). Membrane Model for the G-Protein-Coupled Receptor Rhodopsin: Hydrophobic Interface and Dynamical Structure. Biophysical Journal, 86(4), 2078-2100.
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  PMID: 15041649;PMCID: PMC1304060;Abstract: Rhodopsin is the only member of the pharmacologically important superfamily of G-protein-coupled receptors with a known structure at atomic resolution. A molecular dynamics model of rhodopsin in a POPC phospholipid bilayer was simulated for 15 ns, revealing a conformation significantly different from the recent crystal structures. The structure of the bilayer compared with a protein-free POPC control indicated hydrophobic matching with the nonpolar interface of the receptor, in agreement with deuterium NMR experiments. A new generalized molecular surface method, based on a three-dimensional Voronoi cell construction for atoms with different radii, was developed to quantify cross-sectional area profiles for the protein, lipid acyl chains and headgroups, and water. Thus, it was possible to investigate the bilayer deformation due to curvature of the individual lipid monolayers. Moreover, the generalized molecular surface derived hydrophobic interface allowed benchmarking of the hydropathy sequence analysis, an important structural genomics tool. Five water molecules diffused into internal hydration sites during the simulation, yielding a total of 12 internal waters. The cytoplasmic loops and the C-terminal tail, containing the G-protein recognition and protein sorting sequences, exhibited a high mobility, in marked contrast to the extracellular and transmembrane domains. The proposed functional coupling of the highly conserved ERY motif to the lipid-water interface via the cytoplasmic loops provides insight into lipid effects on G-protein-coupled receptor activation in terms of a flexible surface model, involving the spontaneous monolayer curvature.
• Martinez, G. V., Dykstra, E. M., Lope-Piedrafita, S., & Brown, M. F. (2004). Lanosterol and Cholesterol-Induced Variations in Bilayer Elasticity Probed by 2H NMR Relaxation. Langmuir, 20(4), 1043-1046.
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  PMID: 15803674;Abstract: The influences of lanosterol on lipid bilayers have been compared to those of cholesterol by combining deuterium ( 2H) NMR spin relaxation studies with segmental order parameter measurements. For bilayers of 1,2-diperdeuteriomyristoyl-sn-glycero-3-phosphocholine (DMPC-d 54), the results are consistent with a square-law dependence of the 2H Zeeman relaxation rates (R 1Z) on the corresponding order parameters (S CD). This behavior is indicative of relatively slow order fluctuations, for example, due to quasi-elastic bilayer disturbances. Significant differences are found in the influences of lanosterol versus cholesterol on the microscopic NMR observables; although lanosterol produces smaller order parameters than cholesterol, it leads to larger relaxation rates. By correlating the NMR relaxation behavior with the order parameters, the results are explained by a progressive reduction of the bilayer elasticity, which parallels the biosynthetic pathway from lanosterol to cholesterol.
• Ying, J., Ahn, J. M., Jacobsen, N. E., Brown, M. F., & Hruby, V. J. (2003). NMR Solution Structure of the Glucagon Antagonist [desHis1, desPhe6, Glu9]Glucagon Amide in the Presence of Perdeuterated Dodecylphosphocholine Micelles. Biochemistry, 42, 2825-2835.
• Botelho, A. V., Gibson, N. J., Thurmond, R. L., Wang, Y., & Brown, M. F. (2002). Conformational energetics of rhodopsin modulated by nonlamellar-forming lipids. Biochemistry, 41(20).
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  Rhodopsin is an important example of a G protein-coupled receptor (GPCR) in which 11-cis-retinal is the ligand and acts as an inverse agonist. Photolysis of rhodopsin leads to formation of the activated meta II state from its precursor meta I. Various mechanisms have been proposed to explain how the membrane composition affects the meta I-meta II conformational equilibrium in the visual process. For rod disk membranes and recombinant membranes containing rhodopsin, the lipid properties have been discussed in terms of elastic deformation of the bilayer. Here we have investigated the relation of nonlamellar-forming lipids, such as dioleoylphosphatidylethanolamine (DOPE), together with dioleoylphosphatidylcholine (DOPC), to the photochemistry of membrane-bound rhodopsin. We conducted flash photolysis experiments for bovine rhodopsin recombined with DOPE/DOPC mixtures (0:100 to 75:25) as a function of pH to explore the dependence of the photochemical activity on the monolayer curvature free energy of the membrane. It is well-known that DOPC forms bilayers, whereas DOPE has a propensity to adopt the nonlamellar, reverse hexagonal (H(II)) phase. In the case of neutral DOPE/DOPC recombinants, calculations of the membrane surface pH confirmed that an increase in DOPE favored the meta II state. Moreover, doubling the PE headgroup content versus the native rod membranes substituted for the polyunsaturated, docosahexaenoic acyl chains (22:6 omega 3), suggesting rhodopsin function is associated with a balance of forces within the bilayer. The data are interpreted by applying a flexible surface model, in which the meta II state is stabilized by lipids tending to form the H(II) phase, with a negative spontaneous curvature. A simple theory, based on principles of surface chemistry, for coupling the energetics of membrane proteins to material properties of the bilayer lipids is described. For rhodopsin, the free energy balance of the receptor and the lipids is altered by photoisomerization of retinal and involves curvature stress/strain of the membrane (frustration). A new biophysical principle is introduced: matching of the spontaneous curvature of the lipid bilayer to the mean curvature of the lipid/water interface adjacent to the protein, which balances the lipid/protein solvation energy. In this manner, the thermodynamic driving force for the meta I-meta II conformational change of rhodopsin is tightly controlled by mixtures of nonlamellar-forming lipids having distinctive material properties.
• Brown, M. F., Thurmond, R. L., Dodd, S. W., Otten, D., & Beyer, K. (2002). Elastic deformation of membrane bilayers probed by deuterium NMR relaxation. Journal of the American Chemical Society, 124(28), 8471-8484.
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  PMID: 12105929;Abstract: In deuterium (2H) NMR spectroscopy of fluid lipid bilayers, the average structure is manifested in the segmental order parameters (SCD) of the flexible molecules. The corresponding spin-lattice relaxation rates (R1Z) depend on both the amplitudes and the rates of the segmental fluctuations, and indicate the types of lipid motions. By combining 2H NMR order parameter measurements with relaxation studies, we have obtained a more comprehensive picture of lipids in the liquid-crystalline (Lα) state than formerly possible. Our data suggest that a lipid bilayer constitutes an ordered fluid, in which the phospholipids are grafted to the aqueous interface via their polar headgroups, whereas the fatty acyl chains are in effect liquid hydrocarbon. Studies of 2H-labeled saturated lipids indicate their R1Z rates and SCD order parameters are correlated by a model-free, square-law functional dependence, signifying the presence of relatively slow bilayer fluctuations. A new composite membrane deformation model explains simultaneously the frequency (magnetic field) dependence and the angular anisotropy of the relaxation. The results imply the R1Z rates are due to a broad spectrum of 3-D collective bilayer excitations, together with effective axial rotations of the lipids. For the first time, NMR relaxation studies show that the viscoelastic properties of membrane lipids at megahertz frequencies are modulated by the lipid acyl length (bilayer thickness), polar headgroups (bilayer interfacial area), inclusion of a nonionic detergent (C12E8), and the presence of cholesterol, leading to a range of bilayer softness. Our findings imply the concept of elastic deformation is relevant on lengths approaching the bilayer thickness and less (the mesoscopic scale), and suggest that application of combined R12 and SCD studies of phospholipids can be used as a simple membrane elastometer. Heuristic estimates of the bilayer bending rigidity κ and the area elastic modulus Ka enable comparison to other biophysical studies, involving macroscopic deformation of thin membrane lipid films. Finally, the bilayer softness may be correlated with the lipid diversity of biomembranes, for example, with regard to membrane curvature, repulsive interactions between bilayers, and lipid-protein interactions.
• Brown, M., Brown, M. F., Huber, T., Rajamoorthi, K., Kurze, V. F., & Beyer, K. (2002). Structure of docosahexaenoic acid-containing phospholipid bilayers as studied by 2H NMR and molecular dynamics simulations. Journal of the American Chemical Society, 124(2).
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  Polyunsaturated phospholipids are known to be important with regard to the biological functions of essential fatty acids, for example, involving neural tissues such as the brain and retina. Here we have employed two complementary structural methods for the study of polyunsaturated bilayer lipids, viz. deuterium ((2)H) NMR spectroscopy and molecular dynamics (MD) computer simulations. Our research constitutes one of the first applications of all-atom MD simulations to polyunsaturated lipids containing docosahexaenoic acid (DHA; 22:6 cis-Delta(4,7,10,13,16,19)). Structural features of the highly unsaturated, mixed-chain phospholipid, 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (PDPC), have been studied in the liquid-crystalline (L(alpha)) state and compared to the less unsaturated homolog, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). The (2)H NMR spectra of polyunsaturated bilayers are dramatically different from those of less unsaturated phospholipid bilayers. We show how use of MD simulations can aid in interpreting the complex (2)H NMR spectra of polyunsaturated bilayers, in conjunction with electron density profiles determined from small-angle X-ray diffraction studies. This work clearly demonstrates preferred helical and angle-iron conformations of the polyunsaturated chains in liquid-crystalline bilayers, which favor chain extension while maintaining bilayer flexibility. The presence of relatively long, extended fatty acyl chains may be important for solvating the hydrophobic surfaces of integral membrane proteins, such as rhodopsin. In addition, the polyallylic DHA chains have a tendency to adopt back-bended (hairpin-like) structures, which increase the interfacial area per lipid. Finally, the material properties have been analyzed in terms of the response of the bilayer to mechanical stress. Simulated bilayers of phospholipids containing docosahexaenoic acid were less sensitive to the applied surface tension than were saturated phospholipids, possibly implying a decrease in membrane elasticity (area elastic modulus, bending rigidity). The above features distinguish DHA-containing lipids from saturated or monounsaturated lipids and may be important for their biological modes of action.
• Brown, M., Brown, M. F., Martinez, G. V., Dykstra, E. M., Lope-Piedrafita, S., & Job, C. (2002). NMR elastometry of fluid membranes in the mesoscopic regime. Physical Review E - Statistical, Nonlinear, and Soft Matter Physics, 66(5 Pt 1).
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  In solid-state 2H NMR of fluid lipid bilayers, quasielastic deformations at MHz frequencies are detected as a square-law dependence of the nuclear spin-lattice (R(1Z)) relaxation rates and order parameters (S(CD)). The signature square-law slope is found to decrease progressively with the mole fraction of cholesterol and with the acyl chain length, due to a stiffening of the membrane. The correspondence to thermal vesicle fluctuations and molecular dynamics simulations implies that a broad distribution of modes is present, ranging from the membrane size down to the molecular dimensions.
• Endress, E., Heller, H., Casalta, H., Brown, M. F., & Bayerl, T. M. (2002). Anisotropic motion and molecular dynamics of cholesterol, lanosterol, and ergosterol in lecithin bilayers studied by quasi-elastic neutron scattering. Biochemistry, 41(43), 13078-13086.
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  PMID: 12390036;Abstract: Quasi-elastic neutron scattering (QENS) was employed to study the molecular dynamics of three structurally related sterols, namely, cholesterol, lanosterol, and ergosterol. Oriented bilayers of dipalmitoylphosphatidylcholine (DPPC) were investigated at 40 mol % sterol content and at three temperatures (20, 36, and 50 °C) for two energy resolutions. Data analysis was concentrated on a direct comparison of the out-of-plane and the in-plane high-frequency motions of the three sterols in terms of their rates and amplitudes. The (spatially restricted) diffusive motion of the three sterols in the two directions was characterized by diffusion constants in the range of (5-30) x 10-12 m2 s-1, with a significantly faster rate of diffusion along the membrane normal, resulting in a diffusional anisotropy, Da. At low temperature (20 °C), cholesterol showed the highest value (Da = 4.5), while lanosterol gave the lowest one (Da = 2.0). At high temperature (50 °C), ergosterol diffusion had the highest diffusion anisotropy (Da = 2.0) compared to lanosterol (Da = 1.8) and cholesterol (Da = 1.6). Most interestingly, cholesterol showed at all three temperatures an amplitude of its out-of-plane-motion of 1.0-1.1 nm, more than a factor of 3 higher than measured for the other two sterols. This finding suggests that the short alkyl chain of the cholesterol molecule may cross at high frequency the bilayer midplane, while the other two sterols remain confined within the geometrical limits of each monolayer leaflet. The results provide an example of how slight structural alterations of sterols can affect their molecular dynamics in bilayers, which in turn may be relevant to the membrane micromechanical properties.
• Huber, T., Rajamoorthi, K., Kurze, V. F., Beyer, K., & Brown, M. F. (2002). Structure of docosahexaenoic acid-containing phospholipid bilayers as studied by 2H NMR and molecular dynamics simulations. Journal of the American Chemical Society, 124(2), 298-309.
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  PMID: 11782182;Abstract: Polyunsaturated phospholipids are known to be important with regard to the biological functions of essential fatty acids, for example, involving neural tissues such as the brain and retina. Here we have employed two complementary structural methods for the study of polyunsaturated bilayer lipids, viz. deuterium (2H) NMR spectroscopy and molecular dynamics (MD) computer simulations. Our research constitutes one of the first applications of all-atom MD simulations to polyunsaturated lipids containing docosahexaenoic acid (DHA; 22:6 cis-Δ4,7,10,13,16,19). Structural features of the highly unsaturated, mixedchain phospholipid, 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (PDPC), have been studied in the liquid-crystalline (Lα) state and compared to the less unsaturated homolog, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). The 2H NMR spectra of polyunsaturated bilayers are dramatically different from those of less unsaturated phospholipid bilayers. We show how use of MD simulations can aid in interpreting the complex 2H NMR spectra of polyunsaturated bilayers, in conjunction with electron density profiles determined from small-angle X-ray diffraction studies. This work clearly demonstrates preferred helical and angle-iron conformations of the polyunsaturated chains in liquid-crystalline bilayers, which favor chain extension while maintaining bilayer flexibility. The presence of relatively long, extended fatty acyl chains may be important for solvating the hydrophobic surfaces of integral membrane proteins, such as rhodopsin. In addition, the polyallylic DHA chains have a tendency to adopt back-bended (hairpin-like) structures, which increase the interfacial area per lipid. Finally, the material properties have been analyzed in terms of the response of the bilayer to mechanical stress. Simulated bilayers of phospholipids containing docosahexaenoic acid were less sensitive to the applied surface tension than were saturated phospholipids, possibly implying a decrease in membrane elasticity (area elastic modulus, bending rigidity). The above features distinguish DHA-containing lipids from saturated or monounsaturated lipids and may be important for their biological modes of action.
• Martinez, G. V., Dykstra, E. M., Lope-Piedrafita, S., Job, C., & Brown, M. F. (2002). NMR elastometry of fluid membranes in the mesoscopic regime. Physical Review E - Statistical, Nonlinear, and Soft Matter Physics, 66(5), 050902/1-050902/4.
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  PMID: 12513460;Abstract: In solid-state 2H NMR of fluid lipid bilayers, quasielastic deformations at MHz frequencies are detected as a square-law dependence of the nuclear spin-lattice (R 1z) relaxation rates and order parameters (S CD). The signature square-law slope is found to decrease progressively with the mole fraction of cholesterol and with the acyl chain length, due to a stiffening of the membrane. The correspondence to thermal vesicle fluctuations and molecular dynamics simulations implies that a broad distribution of modes is present, ranging from the membrane size down to the molecular dimensions. © 2002 The American Physical Society.
• Wang, Y., Botelho, A. V., Martinez, G. V., & Brown, M. F. (2002). Electrostatic properties of membrane lipids coupled to metarhodopsin II formation in visual transduction. Journal of the American Chemical Society, 124(26), 7690-7701.
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  Changes in lipid composition have recently been shown to exert appreciable influences on the activities of membrane-bound proteins and peptides. We tested the hypothesis that the conformational states of rhodopsin linked to visual signal transduction are related to biophysical properties of the membrane lipid bilayer. For bovine rhodopsin, the meta I-meta II conformational transition was studied in egg phosphatidylcholine (PC) recombinants versus the native rod outer segment (ROS) membranes by means of flash photolysis. Formation of metarhodopsin II was observed by the change in absorbance at 478 nm after a single actinic flash was delivered to the sample. The meta I/meta II ratio was investigated as a function of both temperature and pH. The data clearly demonstrated thermodynamic reversibility of the transition for both the egg PC recombinants and the native ROS membranes. A significant shift of the apparent pK(a) for the acid-base equilibrium to lower values was evident in the egg PC recombinant, with little meta II produced under physiological conditions. Calculations of the membrane surface pH using a Poisson-Boltzmann model suggested the free energies of the meta I and meta II states were significantly affected by electrostatic properties of the bilayer lipids. In the ROS membranes, phosphatidylserine (PS) is needed for full formation of meta II, in combination with phosphatidylethanolamine (PE) and polyunsaturated docosahexaenoic acid (DHA; 22:6omega3) chains. We propose that the PS surface potential leads to an accumulation of hydronium ions, H(3)O(+), in the electrical double layer, which drive the reaction together with the large negative spontaneous curvature (H(0)) conferred by PE plus DHA chains. The elastic stress/strain of the bilayer arises from an interplay of the approximately zero H(0) from PS and the negative H(0) due to the PE headgroups and polyunsaturated chains. The lipid influences are further explained in terms of matching of the bilayer spontaneous curvature to the curvature at the lipid/rhodopsin interface, as formulated by the Helfrich bending energy. These new findings guide current ideas as to how bilayer properties govern the conformational energetics of integral membrane proteins. Moreover, they yield knowledge of how membrane lipid-protein interactions involving acidic phospholipids such as PS and neutral polyunsaturated DHA chains are implicated in key biological functions such as vision.
• Brown, M. F., Thurmond, R. L., Dodd, S. W., Otten, D., & Beyer, K. (2001). Composite membrane deformation on the mesoscopic length scale. Physical Review E - Statistical, Nonlinear, and Soft Matter Physics, 64(1 I), 010901/1-010901/4.
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  Abstract: Nuclear magnetoresistance (NMR) studies were carried out to investigate a series of phospholipids in the Lα state. The influences of the acyl length, lipid polar head groups, addition of a cosurfactant, and incorporation of cholesterol were determined in terms of the bilayer viscoelastic properties. The results imply that the concept of elastic deformation of membranes is applicable on the mesoscopic length scale, approaching the molecular dimensions, with quasicoherent nodes on the order of the bilayer hydrocarbon thickness and less.
• Petrache, H. I., Salmon, A., & Brown, M. F. (2001). Structural properties of docosahexaenoyl phospholipid bilayers investigated by solid-state 2H NMR spectroscopy. Journal of the American Chemical Society, 123(50), 12611-12622.
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  PMID: 11741426;Abstract: Polyunsaturated lipids in cellular membranes are known to play key roles in such diverse biological processes as vision, neuronal signaling, and apoptosis. One hypothesis is that polyunsaturated lipids are involved in second messenger functions in biological signaling. Another current hypothesis affirms that the functional role of polyunsaturated lipids relies on their ability to modulate physical properties of the lipid bilayer. The present research has employed solid-state 2H NMR spectroscopy to acquire knowledge of the molecular organization and material properties of polyunsaturated lipid bilayers: We report measurements for a homologous series of mixed-chain phosphatidylcholines containing a perdeuterated, saturated acyl chain (n:0) at the sn-1 position, adjacent to docosahexaenoic acid (DHA, 22:6ω3) at the sn-2 position. Measurements have been performed on fluid (Lα)-state multilamellar dispersions as a function of temperature for saturated acyl chain lengths of n = 12, 14, 16, and 18 carbons. The saturated sn-1 chains are therefore used as an intrinsic probe with site-specific resolution of the polyunsaturated bilayer structure. The 2H NMR order parameters as a function of acyl position (order profiles) have been analyzed using a mean-torque potential model for the chain segments; and the results are discussed in comparison with the homologous series of disaturated lipid bilayers. At a given absolute temperature, as the sn-1 acyl length adjacent to the sn-2 DHA chain is greater, the order of the initial chain segments increases, whereas that of the end segments decreases, in marked contrast with the corresponding disaturated series. For the latter, the order of the end segments is practically constant with acyl length, thus revealing a universal chain packing profile. We find that the DHA-containing series, while more complex, is still characterized by a universal chain packing profile, which is shifted relative to the homologous saturated series. Moreover, we show how introduction of DHA chains translates the order profile along the saturated chains, making more disordered states accessible within the bilayer central region. As a result, the area per lipid headgroup is increased as compared to disaturated bilayers. The systematic analysis of the 2H NMR data provides a basis for studies of lipid interactions with integral membrane proteins, for instance in relation to characteristic biological functions of highly unsaturated lipid membranes.
• Otten, D., Brown, M. F., & Beyer, K. (2000). Softening of membrane bilayers by detergents elucidated by deuterium NMR spectroscopy. Journal of Physical Chemistry B, 104(51), 12119-12129.
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  Abstract: Material properties of lipid bilayers were studied on the mesoscopic scale using deuterium nuclear magnetic resonance spectroscopy. The fluid phase of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) was compared with DMPC containing a nonionic detergent as an additive. Order parameter profiles were obtained from the deuterium NMR spectra of DMPC having perdeuterated acyl chains (DMPC-J54). A reduction of the order parameters of DMPC-d54 in the presence of the detergent octaethyleneglycol-mono-/i-dodecyl ether (C12E8) was observed, consistent with an increased configurational freedom of the phospholipid acyl chains. Relaxation rates R1z(i) and R1Q(i) were measured and spectral densities Jm(mω0) where in = 1,2 were calculated from the combined relaxation results. Profiles of the observables, i.e., order parameters, relaxation rates, and spectral densities were interpreted within the framework of a new composite membrane deformation model, which describes characteristic properties of the membrane in terms of a continuum picture. According to this model, the influence of the nonionic detergent (C12E8) on the electroneutral DMPC membrane is to increase the membrane flexibility as manifested by the functional dependence of the R1z(i) and R1Q(i) rates, i.e., the dependence of the spectral densities on the corresponding profiles of the orientational order parameters |SCD(i)|. Within the theoretical framework the increased flexibility of the detergent-containing membranes corresponds to a decrease of the elastic constants for continuum (elastic) deformations of the membrane bilayer. In the case of splay fluctuations the elastic constant and the bilayer thickness are related to the macroscopic bending rigidity, which qualitatively yields a correspondence to studies of macroscopic bending fluctuations thus yielding support for the model. In general, these findings indicate a softening of the membrane bilayer by the presence of a nonionic detergent, which corresponds to a decrease of the elastic constants for continuum deformations of the membrane. © 2000 American Chemical Society.
• Petrache, H. I., Dodd, S. W., & Brown, M. F. (2000). Area per lipid and acyl length distributions in fluid phosphatidylcholines determined by 2H NMR spectroscopy. Biophysical Journal, 79(6), 3172-3192.
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  PMID: 11106622;PMCID: PMC1301193;Abstract: Deuterium (2H) NMR spectroscopy provides detailed information regarding the structural fluctuations of lipid bilayers, including both the equilibrium properties and dynamics. Experimental 2H NMR measurements for the homologous series of 1,2-diacyl-sn-glycero-3-phosphocholines with perdeuterated saturated chains (from C12:0 to C18:0) have been performed on randomly oriented, fully hydrated multilamellar samples. For each lipid, the C-D bond order parameters have been calculated from de-Paked 2H NMR spectra as a function of temperature. The experimental order parameters were analyzed using a mean-torque potential model for the acyl chain segment distributions, and comparison was made with the conventional diamond lattice approach. Statistical mechanical principles were used to relate the measured order parameters to the lipid bilayer structural parameters: the hydrocarbon thickness and the mean interfacial area per lipid. At fixed temperature, the area decreases with increasing acyl length, indicating increased van der Waals attraction for longer lipid chains. However, the main effect of increasing the acyl chain length is on the hydrocarbon thickness rather than on the area per lipid. Expansion coefficients of the structural parameters are reported and interpreted using an empirical free energy function that describes the force balance in fluid bilayers. At the same absolute temperature, the phosphatidylcholine (PC) series exhibits a universal chain packing profile that differs from that of phosphatidylethanolamines (PE). Hence, the lateral packing of phospholipids is more sensitive to the headgroup methylation than to the acyl chain length. A fit to the area per lipid for the PC series using the empirical free energy function shows that the PE area represents a limiting value for the packing of fluid acyl chains.
• Brown, M. F., & Nevzorov, A. A. (1999). 2H-NMR in liquid crystals and membranes. Colloids and Surfaces A, 158(1-2), 281-298.
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  Abstract: Deuterium NMR spectroscopy is widely applicable to studies of the structure and dynamics of molecular solids, liquid crystals, and thin films of membrane lipids. The properties of soft nanomaterials are also accessible on the mesoscopic length scale intermediate between the molecular and bulk dimensions. For membrane lipids in the liquid-crystalline state, rapid axial averaging occurs about the director axis (the membrane normal). One can then relate the profiles of the order parameters |(CD)| of the individual C-2H labeled segments to average bilayer properties. These include the mean area per molecule and projected acyl chain length, the area compressibility modulus, and the radius of curvature for reverse hexagonal (H(II)) phase nanotubes. In addition, measurements of the relaxation rates for Zeeman order, R(1Z), and quadrupolar order, R(1Q), enable one to investigate the mean-squared amplitudes and time-scales of the fluctuations that underlie the thermodynamic properties. A unified interpretation is provided by a composite membrane deformation model, which fits simultaneously the frequency dependence and the angular anisotropy of the R(1Z) and R(1Q) relaxation rates. The results suggest the bilayer dynamics in the MHz regime can be modeled in terms of nematic-like deformations of the membrane hydrocarbon interior, together with axial rotations of the lipid acyl chains. A small contribution from internal segmental motions is found, which implies the bilayer microviscosity is comparable to that of a liquid hydrocarbon. Finally, the 2H-NMR relaxation rates of lipid bilayers containing cholesterol in the liquid-ordered phase suggest a dynamically more rigid bilayer, involving fast axial lipid rotations together with a reduction in collective bilayer deformations. Possible future applications include studies of liquid crystals and thin films of membrane lipids and surfactants, as well as lipid-protein systems.
• Hetzer, M., Gutberiet, T., Brown, M. F., Camps, X., Vostrowsky, O., Schönberger, H., Hirsch, A., & Bayerl, T. M. (1999). Thermotropic Behavior of Lipophilic Derivatized [60]Fullerenes Studied by Deuterium NMR, X-ray Diffraction, and Microcalorimetry. Journal of Physical Chemistry A, 103(5), 637-642.
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  Abstract: The dynamics, structure, and thermotropic behavior of a new class of lipophilic [60]fullerene (C60) derivatives, so-called lipo-fullerenes, have been studied by differential scanning calorimetry (DSC), deuterium nuclear magnetic resonance (2H NMR), and X-ray scattering. The lipo-fullerene studied consists of six pairs of perdeuterated C18 alkyl chains as substituents of six covalently attached methylene groups in octahedral sites. The symmetry of this highly symmetrical hexamethanofullerene is Th We find drastic changes of the molecular arrangement of the lipo-fullerenes induced by temperature. Heating the sample from 20 to 70 °C causes it to undergo two major structural transitions. At 55 °C we observe an exothermic transition from a low-temperature, hard sphere-like packing state of the molecules, with separation distances (6.1 nm) slightly above the maximum diameter of the molecules, to a condensed one. This latter state involves partial intercalation (interdigitation) of the alkyl chains belonging to adjacent molecules and is preceded by partial melting of the chains to accommodate sterically for the (exothermic) interdigitation. The latter allows denser packing with an average separation distance of 4.8 nm. At a temperature of 64 °C, an endothermic melting transition from the interdigitated to a viscous fluidlike state is observed, with an average separation distance of 2.8 nm. Cooling the sample from 70 °C causes a direct transition from the fluid into the low-temperature state with no interdigitation of the chains.
• Kasal, A., Buděšínský, M., Pelnař, J., Bruck, M. A., & Brown, M. F. (1999). Structures and synthesis of 4a-homo-7,19-dinorsteroids, X-ray crystallography and NMR spectroscopy. Collection of Czechoslovak Chemical Communications, 64(12), 2019-2034.
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  Abstract: X-Ray diffraction revealed the absolute configuration of 4aβ-methyl-4a-homo-7,19-dinor-5α,10α-androstane-3,17-dione. Detailed NMR analysis suggested that the 5α configuration existed in the starting material, 3β-acetoxy-4a-methylidene-4a-homo-7,19-dinor-5α-androst-9-en-17-one, and related compounds. Thus 5-methyl-5β-estr-9-ene derivatives with a leaving group in position 6β were found to react with nucleophiles to form rearranged 4a-homo-7,19-dinorandrostane derivatives with a 5α configuration.
• Moltke, S., Wallat, I., Sakai, N., Nakanishi, K., Brown, M. F., & Heyn, M. P. (1999). The angles between the C1-, C5-, and C9-methyl bonds of the retinylidene chromophore and the membrane normal increase in the M intermediate of bacteriorhodopsin: Direct determination with solid-state 2H NMR. Biochemistry, 38(36), 11762-11772.
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  PMID: 10512633;Abstract: The orientations of three methyl bonds of the retinylidene chromophore of bacteriorhodopsin were investigated in the M photointermediate using deuterium solid-state NMR (2H NMR). In this key intermediate, the chromophore has a 13-cis, 15-anti conformation and a deprotonated Schiff base. Purple membranes containing wild-type or mutant D96A bacteriorhodopsin were regenerated with retinals specifically deuterated in the methyl groups of either carbon C1 or C5 of the β-ionone ring or carbon C9 of the polyene chain. Oriented hydrated films were formed by drying concentrated suspensions on glass plates at 86% relative humidity. The lifetime of the M state was increased in the wild-type samples by applying a guanidine hydrochloride solution at pH 9.5 and in the D96A sample by raising the pH. 2H NMR experiments were performed on the dark-adapted ground state (a 2:1 mixture of 13-cis, 15-syn and all-trans, 15-anti chromophores), the cryotrapped light-adapted state (all-trans, 15-anti), and the cryotrapped M intermediate (13-cis, 15-anti) at -50 °C. Bacteriorhodopsin was first completely converted to M under steady illumination of the hydrated films at +5 °C and then rapidly cooled to -50 °C in the dark. From a tilt series of the oriented sample in the magnetic field and an analysis of the 2H NMR line shapes, the angles between the individual C-CD3 bonds and the membrane normal could be determined even in the presence of a substantial degree of orientational disorder. While only minor differences were detected between dark- and light-adapted states, all three angles increase in the M state. This is consistent with an upward movement of the C5-C13 part of the polyene chain toward the cytoplasmic surface or with increased torsional strain. The C9-CD3 bond shows the largest orientational change of 7°in M. This reorientation of the chromophore in the binding pocket provides direct structural support for previous suggestions (based on spectroscopic evidence) for a steric interaction in M between the C9-methyl group and Trp 182 in helix F.
• Nevzorov, A. A., Moltke, S., Heyn, M. P., & Brown, M. F. (1999). Solid-state NMR line shapes of uniaxially oriented immobile systems. Journal of the American Chemical Society, 121(33), 7636-7643.
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  Abstract: The problem of simulating the spectral line shapes of aligned immobile samples arises in solid-state NMR of various biological systems, including integral membrane proteins and peptides, receptor-bound ligands, and macroscopically oriented DNA fibers. An important issue with regard to the extraction of structural information is the correct treatment of the distribution of local symmetry axes relative to the average alignment axis (mosaic spread). Previous formulations have not considered explicitly the three-dimensional uniaxial character of the local axis disorder. Rather, the mosaic spread has been treated simply by convoluting the theoretical line shape function with an effectively two-dimensional distribution of the local symmetry axes. Here a closed-form line shape expression is derived for an axially symmetric distribution of bond orientations, which includes the uniaxial distribution of the local symmetry axis about the average alignment axis. As an illustration, the influences of the bond orientation and the degree of mosaic spread on deuterium (2H) NMR line shapes are investigated. The closed-form solution in terms of elliptic integrals gives virtually identical results to those of an alternative numerical Monte Carlo line shape simulation method. The derived line shape function yields the correct powder- type limit, and has been tested by simulating a tilt series of 2H NMR spectra of purple membranes containing bacteriorhodopsin with a specifically deuterated 1R methyl group in the retinal ring. The probability distribution for the bond orientations derived herein can be of potential interest for solid-state NMR spectroscopy of aligned biomolecules involving dipolar, quadropolar, and chemical shift interactions, such as integral membrane proteins and peptides.
• Salamon, Z., Brown, M. F., & Tollin, G. (1999). Plasmon resonance spectroscopy: Probing molecular interactions within membranes. Trends in Biochemical Sciences, 24(6), 213-219.
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  PMID: 10366845;Abstract: Surface plasmon resonance (SPR) has become a popular method for investigating biomolecular interactions. A new variant of this technique, coupled plasmon-waveguide resonance (CPWR) spectroscopy, allows the characterization of anisotropic biological membranes. Plasmon resonance can therefore be used to study the molecular events involved in a wide variety of membrane processes, including energy conversion and signal transduction.
• Trouard, T. P., Nevzorov, A. A., Alam, T. M., Job, C., Zajicek, J., & Brown, M. F. (1999). Influence of Cholesterol on Dynamics of Dimyristoylphosphatidylcholine Bilayers as Studied by Deuterium NMR Relaxation. The Journal of Chemical Physics, 110, 8802-8818.
• Molkte, S., Nevzorov, A. A., Sakai, N., Wallat, I., Job, C., Nakanishi, K., Heyn, M. P., & Brown, M. F. (1998). Chromophore orientation in bacteriorhodopsin determined from the angular dependence of deuterium nuclear magnetic resonance spectra of oriented purple membranes. Biochemistry, 37(34), 11821-11835.
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  PMID: 9718305;Abstract: The orientation of prosthetic groups in membrane proteins is of considerable importance in understanding their functional role in energy conversion, signal transduction, and ion transport. In this work, the orientation of the retinylidene chromophore of bacteriorhodopsin (bR) was investigated using 2H NMR spectroscopy. Bacteriorhodopsin was regenerated with all-trans-retinal stereospecifically deuterated in one of the geminal methyl groups on C1 of the cyclohexene ring. A highly oriented sample, which is needed to obtain individual bond orientations from 2H NMR, was prepared by forming hydrated lamellar films of purple membranes on glass slides. A Monte Carlo method was developed to accurately simulate the 2H NMR line shape due to the distribution of bond angles and the orientational disorder of the membranes. The number of free parameters in the line shape simulation was reduced by independent measurements of the intrinsic line width (1.6 kHz from T(2e experiments) and the effective quadrupolar coupling constant (38.8- 39.8 kHz from analysis of the line shape of a powder-type sample). The angle between the C1-(1R)-1-CD3 bond and the purple membrane normal was determined with high accuracy from the simultaneous analysis of a series of 2H NMR spectra recorded at different inclinations of the uniaxially oriented sample in the magnetic field at 20 and -50 °C. The value of 68.7 ± 2.0°in dark-adapted bR was used, together with the previously determined angle of the C5-CD3 bond, to calculate the possible orientations of the cyclohexene ring in the membrane. The solutions obtained from 2H NMR were then combined with additional constraints from linear dichroism and electron cryomicroscopy to obtain the allowed orientations of retinal in the noncentrosymmetric membrane structure. The combined data indicate that the methyl groups on the polyene chain point toward the cytoplasmic side of the membrane and the N-H bond of the Schiff base to the extracellular side, i.e., toward the side of proton release in the pump pathway.
• Nevzorov, A. A., Moltke, S., & Brown, M. F. (1998). Structure of the A-form and B-form of DNA from deuterium NMR line shape simulation. Journal of the American Chemical Society, 120(19), 4798-4805.
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  Abstract: Simulation of experimental solid-state deuterium (2H) NMR spectra of nucleic acid fibers allows one to deduce important microscopic information about the orientation of the base pairs and the helix axis disorder. However, existing interpretations of the 2H NMR spectra of Na-DNA at low humidity are not in complete agreement with the X-ray results. Here we have successfully explained 2H NMR spectra of oriented films of both Li-DNA and Na-DNA with the purine bases specifically deuterated at position C8. The transformation of the coupling tensor from the principal axis system to the laboratory frame has been expanded into four subtransformations, including the crystallographically defined base plane tilt and roll angles. Alternative treatments in terms of noncollective or collective helix axis disorder are considered, and the appropriate powder-pattern limits are recovered. The 2H NMR spectral line shapes have been calculated by using the Monte Carlo method, i.e., by randomly sampling over the static uniaxial distributions of the base pairs and helix axes. The results of the simulations and the structural parameters are in excellent agreement with X-ray diffraction studies, which indicate the presence of either the A-form or B-form of DNA under the given experimental conditions. Only a static distribution of base pairs is needed to account for the spectral line shapes on the 2H NMR time scale, whereas the effects of faster librational motions are contained in the intrinsic line widths and the effective coupling constants. The present 2H NMR approach can aid in developing a more comprehensive picture of DNA conformation and dynamics as an adjunct to X-ray crystallography, fluorescence depolarization, and light-scattering methods, and moreover may prove useful in studies of protein-nucleic acid interactions.
• Nevzorov, A. A., Trouard, T. P., & Brown, M. F. (1998). Lipid Bilayer Dynamics from Simultaneous Analysis of Orientation and Frequency Dependence of Deuterium Spin-Lattice and Quadrupolar Order Relaxation. Physical Review E - Statistical, Nonlinear, and Soft Matter Physics, 58, 2259-2281.
• Brown, M. F. (1997). Influence of Nonlamellar-Forming Lipids on Rhodopsin. Current Topics in Membranes, 44(C), 285-356.
• Brown, M. F., Nevzorov, A. A., & Trouard, T. P. (1997). Correlation Functions for Lipid Membrane Fluctuations Obtained from NMR Spectroscopy. Physical Review E - Statistical, Nonlinear, and Soft Matter Physics, 55, 3276-3282.
• Nevzorov, A. A., & Brown, M. F. (1997). Dynamics of lipid bilayers from comparative analysis of 2H and 13C nuclear magnetic resonance relaxation data as a function of frequency and temperature. The Journal of Chemical Physics, 107(23), 10288-10310.
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  Abstract: Analysis of the nuclear spin relaxation rates of lipid membranes provides a powerful means of studying the dynamics of these important biological representatives of soft matter. Here, temperature- and frequency-dependent 2H and 13C nuclear magnetic resonance (NMR) relaxation rates for vesicles and multilamellar dispersions of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) in the liquid-crystalline state have been fitted simultaneously to various dynamic models for different positions of the acyl chains. The data include 2H R1z rates (Zeeman order of electric quadrupolar interaction) acquired at 12 external magnetic field strengths from 0.382 to 14.6 T, corresponding to a frequency range from ωD/2π=2.50-95.3 MHz; and 2H R1Q rates (quadrupolar order of electric quadrupolar interaction) at 15.3, 46.1, and 76.8 MHz. Moreover, 13C R1z data (Zeeman order of magnetic dipolar interaction) for DMPC are included at six magnetic field strengths, ranging from 1.40 to 17.6 T, thereby enabling extension of the frequency range to effectively (ωC+ωH)/2π=938.7 MHz. Use of the generalized approach allows formulation of noncollective segmental and molecular diffusion models, as well as collective director fluctuation models, which were tested by fitting the 2H R1Z data at different frequencies and temperatures (30 °C and 50 °C). The corresponding 13C relaxation rates were predicted theoretically and compared to experiment, thus allowing one to unify the 13C and 2H NMR data for bilayer lipids in the fluid state. A further new aspect is that the spectral densities of motion have been explicitly calculated from the 2H R1Z and R1Q data at 40 °C. We conclude that the relaxation in fluid membrane bilayers is governed predominantly by relatively slow motions, which modulate the residual coupling remaining from faster local motions (order fluctuations). Only the molecular diffusion model, including an additional slow motional process, and the membrane deformation model describing three-dimensional collective fluctuations fit the 2H NMR data and predict the 13C NMR data in the MHz range. Orientational correlation functions have been calculated, which emphasizes the importance of NMR relaxation as a unique tool for investigating the dynamics of lipid bilayers and biological membranes. © 1997 American Institute of Physics.
• Schroeder, T. P., Job, C., Brown, M. F., Glass, R. S., You, N., & Block, E. (1997). 1H-{125Te} Indirect Detection in Nuclear Magnetic Resonance Spectra of Organotellurium Compounds. Magnetic Resonance in Chemistry, 35, 752-756.
• Job, C., Zajicek, J., & Brown, M. F. (1996). Fast field-cycling nuclear magnetic resonance spectrometer. Review of Scientific Instruments, 67(6), 2113-2122.
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  Abstract: We describe here the design and construction of a modern, state-of-the-art nuclear magnetic resonance (NMR) field-cycling instrument. Fourier transform NMR spectra of both liquid and solid samples can be measured, and spin-lattice relaxation times (T1Z) investigated over a broad range of magnetic field strengths ranging from 0 to 2 T. The instrument is based upon an existing personal computer-based NMR spectrometer [C. Job, R. M. Pearson, and M. F. Brown, Rev. Sci. Instrum. 65, 3354 (1994)] which has been expanded into a fully computer-controlled field-cycling instrument. The magnetic field cycling is accomplished electronically by utilizing fast switching thyristors and a storage capacitor based on the Redfield energy storage concept. Unique aspects of the design include the field-cycling magnet, which can reach fields as high as 2 T; the personal computer-based NMR spectrometer and associated waveform electronics; and the use of a commercially available pulse width modulation switching current amplifier, having low internal power dissipation and a fast current settling time. Using this new technology T1Z relaxation times as short as 1 ms can be readily measured. © 1996 American Institute of Physics.
• Salamon, Z., Wang, Y., Soulages, J. L., Brown, M. F., & Tollin, G. (1996). Surface plasmon resonance spectroscopy studies of membrane proteins: Transducin binding and activation by rhodopsin monitored in thin membrane films. Biophysical Journal, 71(1), 283-294.
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  PMID: 8804611;PMCID: PMC1233479;Abstract: Surface plasmon resonance (SPR) spectroscopy can provide useful information regarding average structural properties of membrane films supported on planar solid substrates. Here we have used SPR spectroscopy for the first time to monitor the binding and activation of G-protein (transducin or G(t)) by bovine rhodopsin incorporated into an egg phosphatidylcholine bilayer deposited on a silver film. Rhodopsin incorporation into the membrane, performed by dilution of a detergent solution of the protein, proceeds in a saturable manner. Before photolysis, the SPR data show that G(t) binds tightly (K(eq) ≃ 60 nM) and with positive cooperativity to rhodopsin in the lipid layer to form a closely packed film. A simple multilayer model yields a calculated average thickness of about 57 .Å, in good agreement with the structure of G(t). The data also demonstrate that G(t) binding saturates at a G(t)/rhodopsin ratio of approximately 0.6. Moreover, upon visible light irradiation, characteristic changes occur in the SPR spectrum, which can be modeled by a 6 Å increase in the average thickness of the lipid/protein film caused by formation of metarhodopsin II (MII). Upon subsequent addition of GTP, further SPR spectral changes are induced. These are interpreted as resulting from dissociation of the α- subunit of G(t), formation of new MII-G(t) complexes, and possible conformational changes of G(t) as a consequence of complex formation. The above results clearly demonstrate the ability of SPR spectroscopy to monitor interactions among the proteins associated with signal transduction in membrane-bound systems.
• Schroeder, T. B., Job, C., Brown, M. F., & Glass, R. S. (1995). Indirect Detection of Selenium-77 in Nuclear Magnetic Resonance Spectra of Organoselenium Compounds. Magnetic Resonance in Chemistry, 33, 191-195.
• Zajicek, J., Ellena, J. F., Williams, G. D., Khadim, M., & Brown, M. F. (1995). Molecular Dynamics of Vesicles of Unsaturated Phosphatidylcholines Studied by 13C NMR Spin-Lattice Relaxation. Collection of Czechoslovak Chemical Communications, 60, 719-735.
• Brown, M. F. (1994). Modulation of rhodopsin function by properties of the membrane bilayer. Chemistry and Physics of Lipids, 73(1-2), 159-180.
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  PMID: 8001180;Abstract: A prevalent model for the function of rhodopsin centers on the metarhodopsin I (MI) to metarhodopsin II (MII) conformational transition as the triggering event for the visual process. Flash photolysis techniques enable one to determine the [MII]/[MI] ratio for rhodopsin in various recombinant membranes, and thus investigate the roles of the phospholipid head groups and the lipid acyl chains systematically. The results obtained to date clearly show that the pK for the acid-base MI-MII equilibrium of rhodopsin is modulated by the lipid environment. In bilayers of phosphatidylcholines the MI-MII equilibrium is shifted to the left; whereas in the native rod outer segment membranes it is shifted to the right, i.e., at neutral pH near physiological temperature. The lipid mixtures sufficient to yield full photochemical function of rhodopsin include a native-like head group composition, viz, comprising phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS), in combination with polyunsaturated docosahexaenoic acid (DHA; 22:6ω3) chains. Yet such a native-like lipid mixture is not necessary for the MI-MII conformational transition of rhodopsin; one can substitute other lipid compositions having similar properties. The MI-MII transition is favored by relatively small head groups which produce a condensed bilayer surface, viz, a comparatively small interfacial area as in the case of PE, together with bulky acyl chains such as DHA which prefer a relatively large cross sectional area. The resulting force imbalance across the layer gives rise to a curvature elastic stress of the lipid/water interface, such that the lipid mixtures yielding native-like behavior form reverse hexagonal (HII) phases at slightly higher temperatures. A relatively unstable membrane is needed; lipids tending to form the lamellar phase do not support full native-like photochemical function of rhodopsin. Thus chemically specific properties of the various lipids are not required, but rather average or material properties of the entire assembly, which may involve the curvature free energy of the membrane-lipid water interface. These findings reveal that the membrane lipid bilayer has a direct influence on the energetics of the conformational states of rhodopsin in visual excitation. © 1994.
• Job, C., Pearson, R. M., & Brown, M. F. (1994). A personal computer-based nuclear magnetic resonance spectrometer. Review of Scientific Instruments, 65(11), 3354-3362.
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  Abstract: Nuclear magnetic resonance (NMR) spectroscopy using personal computer-based hardware has the potential of enabling the application of NMR methods to fields where conventional state of the art equipment is either impractical or too costly. With such a strategy for data acquisition and processing, disciplines including civil engineering, agriculture, geology, archaeology, and others have the possibility of utilizing magnetic resonance techniques within the laboratory or conducting applications directly in the field. Another aspect is the possibility of utilizing existing NMR magnets which may be in good condition but unused because of outdated or nonrepairable electronics. Moreover, NMR applications based on personal computer technology may open up teaching possibilities at the college or even secondary school level. The goal of developing such a personal computer (PC)-based NMR standard is facilitated by existing technologies including logic cell arrays, direct digital frequency synthesis, use of PC-based electrical engineering software tools to fabricate electronic circuits, and the use of permanent magnets based on neodymium-iron-boron alloy. Utilizing such an approach, we have been able to place essentially an entire NMR spectrometer console on two printed circuit boards, with the exception of the receiver and radio frequency power amplifier. Future upgrades to include the deuterium lock and the decoupler unit are readily envisioned. The continued development of such PC-based NMR spectrometers is expected to benefit from the fast growing, practical, and low cost personal computer market.
• Salamon, Z., Wang, Y., Brown, M. F., MacLeod, A., & Tollin, G. (1994). Conformational Changes in Rhodopsin Probed by Surface Plasmon Resonance Spectroscopy. Biochemistry, 33, 13706-13711.
• Thurmond, R. L., Otten, D., Brown, M. F., & Beyer, K. (1994). Structure and packing of phosphatidylcholines in lamellar and hexagonal liquid-crystalline mixtures with a nonionic detergent: A wide-line deuterium and phosphorus-31 NMR study. Journal of Physical Chemistry, 98(3), 972-983.
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  Abstract: Lamellar and hexagonal (HI) liquid-crystalline mixtures of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) with the nonionic detergent octaethyleneglycol mono-n-dodecyl ether (C12E8) have been studied by solid-state 2H and 31P NMR spectral techniques. The HI phase structure is considered as a useful model for small mixed detergent/phospholipid micelles. Formation of such mixed micelles is an obligatory step in the investigation of biological membranes and membrane proteins. Both the phospholipid 31P NMR chemical shift anisotropy and the 2H NMR quadrupole splittings can be observed in the hexagonal mesophase, in contrast to micelles where motional averaging precludes the direct observation of residual chemical shift or quadrupolar tensors. The 2H NMR spectra were evaluated in terms of carbon-deuterium bond order parameter profiles. The order profiles obtained in the lamellar detergent/phospholipid mixtures were significantly different from the corresponding profiles in the mixed detergent/phospholipid HI phase. In the lamellar phase, an increasing proportion of C12E8 led to a significant reduction in the absolute magnitude of the individual order parameters, whereas in the HI phase the order parameters remained relatively constant over a broad range of detergent/ phospholipid molar ratios. It was also shown by 31P and 2H NMR that the interfacial segments of the phospholipid (the glycerol backbone and the headgroup dipole) assume an almost identical conformation in the lamellar and in the HI phase. Average chain lengths and mean cross-sectional areas were derived from the order parameter profiles and interpreted in terms of geometrical properties of the hexagonal and lamellar phase aggregates. Furthermore, measurements at different temperatures yielded an estimate of thermal expansion coefficients of the phospholipid acyl chains in the different phases. It is concluded that addition of detergent to lamellar phospholipids results in increasing packing constraints which are eventually relieved by a lamellar → HI transition. These packing constraints are discussed in terms of the molecular shape concept and with reference to statistical mechanical models of chain packing in bilayers, HI-structures, and small micelles. © 1994 American Chemical Society.
• Trouard, T. P., Alam, T. M., Job, C., & Brown, M. F. (1994). Angular Dependence of Deuterium Spin-Lattice Relaxation of Dilaurylphosphatidylcholine in the Liquid-Crystalline Phase. The Journal of Chemical Physics, 101, 5229-5261.
• Brown, M. F. (1993). Lipid headgroup and acyl chain composition modulate the MI-MII equilibrium of rhodopsin in recombinant membranes. Biochemistry, 32(9), 2438-2454.
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  PMID: 8443184;Abstract: A current paradigm for visual function centers on the metarhodopsin I (MI) to metarhodopsin II (MII) conformational transition as the trigger for an intracellular enzyme cascade leading to excitation of the retinal rod. We investigated the influences of the membrane lipid composition on this key triggering event in visual signal transduction using flash photolysis techniques. Bovine rhodopsin was combined with various phospholipids to form membrane recombinants in which the lipid acyl chain composition was held constant at that of egg phosphatidylcholine (PC), while the identity of the lipid headgroups was varied. The ratio of MII/MI produced in these recombinants by an actinic flash at 28°C was studied as a function of pH. The results were compared to the photochemical function observed for rhodopsin in native retinal rod outer segment (ROS) membranes, in total native ROS lipid recombinants, and in dimyristoylphosphatidylcholine (DMPC) recombinants. In membrane recombinants incorporating lipids derived from egg PC, as well as in the total ROS lipids control and the native ROS disk membranes, MI and MII were found to coexist in a pH-dependent, acid-base equilibrium on the millisecond time scale. The recombinants of rhodopsin with egg PC, either alone or in combination with egg PC-derived phosphatidylethanolamine (PE) or phosphatidylserine (PS), exhibited substantially reduced photochemical activity at pH 7.0. However, all recombinants comprising phospholipids with unsaturated acyl chains were capable of full native-like MII production at pH 5.0, confirming previous results [Gibson, N. J., & Brown, M. F. (1990) Biochem. Biophys. Res. Commun. 169, 1028-1034]. It follows that energetic constraints on the MI and MII states imposed by egg PC-derived acyl chains can be offset by increased activity of H+ ions. The data reveal that the major effect of the membrane lipid composition is to alter the apparent pK for the MI-MII conformational equilibrium of rhodopsin [Gibson, N. J., & Brown, M. F. (1991) Biochem. Biophys. Res. Commun. 176, 915-921]. Recombinants containing only phosphocholine headgroups exhibited the lowest apparent pK values, whereas the presence of either 50 mol % PE or 15 mol % PS increased the apparent pK. The inability to obtain full native-like function in recombinants having egg PC-derived chains and a native-like headgroup composition indicates a significant role of the polyunsaturated docosahexaenoic acid (DHA) chains (22:6ω3) of the native retinal rod membrane lipids. Temperature studies of the MI-MII transition enabled an investigation of lipid influences on the thermodynamic parameters of a membrane protein conformational change linked directly to function. The changes in thermodynamic state variables suggest that rhodopsin may be partially unfolded in the MII state, leading to exposure of recognition sites for the signal transducing G protein. Finally, the results are discussed in terms of properties of the membrane lipid bilayer, including the influences of bilayer electrostatics as well as bulk material properties associated with the protein/lipid and lipid/water interfaces. Relatively small changes due to lateral and/or curvature stresses involving the lipid/water interface are sufficient to explain the free energy shifts for the MI-MII transition among the recombinants. The combination of PE headgroups together with bulky DHA chains in the native retinal rod lipids promotes formation of nonlamellar phases; one possibility is that the curvature free energy of the membrane- lipid/water interface is involved. These findings indicate that the membrane lipid composition influences directly the photochemical activity of rhodopsin, thereby implicating properties of the membrane lipid bilayer in the molecular mechanism of the visual process. © 1993 American Chemical Society.
• Thurmond, R. L., Lindblom, G., & Brown, M. F. (1993). Curvature, order, and dynamics of lipid hexagonal phases studied by deuterium NMR spectroscopy. Biochemistry, 32(20), 5394-5410.
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  PMID: 8499443;Abstract: Solid-state deuterium (2H) NMR spectroscopy enables one to study both equilibrium and dynamical properties of membrane constituents at the molecular level and can yield significant insights regarding the organization of non-bilayer lipid aggregates. We have investigated a representative unsaturated phosphatidylethanolamine, viz., 1-perdeuteriopalmitoyl-1-linoleoyl-sn-glycero-S-phosphoethanolamine, PLPE-d31, in the lamellar, or Lα, phase and the reversed hexagonal, or HII, phase. Phosphorus-31 (31P) NMR studies of PLPE-d31 in the HII phase revealed that the chemical shift anisotropy of the phosphoethanolamine head groups, Δσ, was scaled by the expected geometrical factor of -1/2 relative to the lamellar state. However, we found the occurrence of a further reduction in the 2H NMR quadrupolar splittings, ΔνQ, of the 2H-labeled palmitoyl acyl chain segments. These observations point toward the role of interfacial curvature with regard to properties of reverse hexagonal phase lipids, and indicate that the pivotal position or neutral surface of approximately constant area may lie near the glycerol or polar head group region. Variations in the acyl chain packing due to curvature of the aqueous interface yield significant differences in the segmental order profiles as determined by 2H NMR spectroscopy. The latter reflect the local orientational order of the acyl chains and can be used together with simple statistical theories to extract positional or structural information. Average projected acyl chain lengths and mean interfacial or cross-sectional areas for PLPE-d31 in the different phases have been calculated. In addition, we describe a new means of estimating the radius of curvature of HII phase lipid aggregates utilizing 2H NMR spectroscopy, which is based on the difference between the lamellar and hexagonal phase order profiles. Here the radius of curvature, Rc, is defined as the distance from the center of the water core to the lipid/water interface, near the carbonyl segments of the acyl chains, giving Rc = 25.4-28.1 Å for PLPE-d31 in the HII phase at 60°C. This value is in good agreement with previous X-ray diffraction studies of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). Alternatively, the data yield for the radius of the central water core that Rw = 17.8-20.5 Å at 60°C. The differences in geometry also lead to higher quadrupolar echo relaxation rates (R2e) for the lipid acyl segments closest to the aqueous interface in the HII versus the Lα phase. We propose that this enhancement is due to an additional relaxation mechanism found in the hexagonal phases, namely, translational diffusion of lipids about the cylinder axes. For comparison, the normal hexagonal (HI) and lamellar (Lα) phases of a lyotropic system comprising perdeuterated potassium laurate were also studied. This research indicates clearly that the packing and dynamical properties of the acyl chains of phospholipids depend on the curvature of the aqueous interface and, thus, the aggregate geometry. The latter is related to the average shape of lipids in their respective phases and to the curvature free energy, which in the planar state may influence protein-mediated functions of membranes. © 1993 American Chemical Society.
• Alexander, A. L., Pytlewski, V. T., Brown, M. F., & Gmitro, A. F. (1992). Detection of atherosclerosis via magnetic resonance imaging. Proceedings of SPIE - The International Society for Optical Engineering, 1642, 26-33.
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  Abstract: Magnetic resonance imaging (MRI) of atherosclerotic lipids using a stimulated-echo diffusion- weighted (STED) sequence is demonstrated. The STED sequence exploits the large difference in diffusion between lipid (primarily cholesteryl ester) and water. The optimization of the STED sequence is discussed. The results of lipid imaging are corroborated with nuclear magnetic resonance (NMR) spectroscopy. This technique is non-invasive, and therefore, it is potentially useful in following the progression of the disease in animal models and in humans.
• Barry, J. A., Lamparski, H., Shyamsunder, E., Osterberg, F., Cerne, J., Brown, M. F., & O'Brien, D. F. (1992). 31P NMR and X-ray diffraction study of the effect of photopolymerization on lipid polymorphism. Biochemistry, 31(41), 10114-10120.
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  PMID: 1390768;Abstract: It was recently shown that oligolamellar vesicles of 3:1 mixtures of dioleoylphosphatidylethanolamine (DOPE) and the photopolymerizable lipid 1,2-bis[10-(2′,4′-hexadienoyloxy)decanoyl]-sn-glycero-3- phosphocholine (SorbPC) are destabilized by polymerization of the SorbPC [Lamparski, H., Liman, U., Frankel, D. A., Barry, J. A., Ramaswami, V., Brown, M. F., & O'Brien, D. F. (1992) Biochemistry 31, 685-694]. The current work describes the polymorphic phase behavior of these mixtures in extended bilayers, as studied by 31P NMR spectroscopy and X-ray diffraction. In the NMR experiments, samples with varying degrees of polymerization were slowly raised in temperature, with spectra acquired every 2.5-10°C. In the unpolymerized mixiture, and in those photopolymerized samples where the monomeric SorbPC was decreased by 33% and 51%, an isotropic signal grew progressively until no signal from the lamellar liquid-crystalline (Lα) phase remained. In the highly polymerized sample with a 90% loss of monomeric SorbPC, less than 20% of the lipids underwent this transition. In none of the samples was an inverted hexagonal phase (HII) observed, under conditions of slow heating to almost 100°C. The X-ray diffraction studies indicated that samples which exhibit the isotropic NMR signal corresponded to a structure exhibiting no well-defined crystalline order, which upon thermal cycling became an inverted cubic phase belonging to either the Pn3m or Pn3 space groups. The temperature of the transition to the cubic precursor decreased as the extent of polymerization increased, demonstrating that photopolymerization of these lipid bilayers can significantly alter the composition and thermotropic phase behavior of the mixture. © 1992 American Chemical Society.
• Jansson, M., Thurmond, R. L., Barry, J. A., & Brown, M. F. (1992). Deuterium NMR study of intermolecular interactions in lamellar phases containing palmitoyllysophosphatidylcholine. Journal of Physical Chemistry, 96(23), 9532-9544.
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  Abstract: Theoretical models of phospholipid systems have indicated that both intramolecular and intermolecular forces are important in governing their acyl chain order. Knowledge of the nature and magnitude of these interactions is central to understanding the balance of forces present in lipid lamellar phases, which in turn is related to their microscopic and macroscopic behavior. It is possible to explore the contribution of iniermolecular interactions using lipid systems with the same headgroup and acyl chain identity by variation of the ratio of the headgroups to acyl chains. In this paper, deuterium (2H) NMR spectroscopy has been used to gain information on the orientational order of an acyl chain perdeuterated lipid, 1-perdeuteriopalmitoyl-sn-glycero-3-phosphocholine (PaLPC-d31), in various molecular environments. The orientational order of PaLPC-d31, was studied in four different lamellar phases, including pure PaLPC-d31 (containing 10 wt % H2O), dipalmitoylphosphatidylcholine/PaLPC-d31 (3:1), palmitic acid/PaLPC-d31 (1:1), and cholesterol/PaLPC-d31 (1:1) (each containing 50 wt % H2O) 2H NMR spectra were obtained for the low-temperature and liquid-crystalline (Lα) states of each of these mixtures. In the low-temperature state, the first three systems yielded 2H NMR spectra characteristic of all-trans chains undergoing axial diffusion, with the methyl groups rotating about their C3 axes. The molecular order, as judged by the presence of spectral discontinuities and moment analysis, was found to be almost identical in the low-temperature phases. A different behavior was observed for the cholesterol/PaLPC-d31 (1:1) sample in that the maximum splitting was close to the all-trans rotating value, with a profile of quadrupolar splittings due to increased disorder near the chain ends. The first three systems underwent order-disorder phase transitions near the same midpoint temperature (range of Tm values 40-48°C), whereas the chotesterol/PaLPC-d31 (1:1) sample did not display a transition over the temperature range studied. In the Lα phase, where order profiles were determined as a function of acyl chain segment position, the segmental ordering differed significantly among the samples. The differences were interpreted using a simple diamond lattice model for the acyl chain configurational statistics, as a means of comparing the effective lengths, 〈L〉, projected along the bilayer normal and estimated chain cross-sectional areas, 〈A〉, of PaLPC-d31 in the various mixtures. The derived values of 〈L〉 and 〈A〉 can be understood qualitatively in terms of average packing parameters related to the balance of forces in the headgroup and acyl chain regions, or alternatively the curvature free energy of the membrane lipid-water interface. In lamellar phases of pure PaLPC-d31 the curvature stress is potentially large, and interdigitation of the acyl chains of the apposed monolayers may occur. However, in mixtures of PaLPC-d31 with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), the curvature elastic stress is apparently relieved by an increase in the cross-sectional acyl chain area, 〈A〉, i.e. corresponding to an increase in configurational freedom. The data were also compared to the results of statistical theories to yield additional knowledge of the intermolecular forces. These studies indicate how the segmental ordering reflects lute/molecular interactions within a given lamellar phase. Average properties of the entire system such as average cross-sectional area accessible to each acyl chain relative to the headgroup area can be modulated by these interactions. Such mfermolecular interactions may be related to the presence of lipid diversity in biological membranes. © 1992 American Chemical Society.
• Lamparski, H., Liman, U., Barry, J. A., Frankel, D. A., Ramaswami, V., Brown, M. F., & O'Brien, D. F. (1992). Photoinduced destabilization of liposomes. Biochemistry, 31(3), 685-694.
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  PMID: 1731924;Abstract: The stability of two-component liposomes composed of the polymerizable 1,2-bis-[10-(2′,4′-hexadienoyloxy)decanoyl]-sn-glycero-3- phosphatidylcholine (SorbPC) and either a phosphatidylethanolamine (PE) or a phosphatidylcholine (PC) were examined via fluorescence leakage assays. Ultraviolet light exposure of SorbPC-containing liposomes forms poly-SorbPC, which phase separates from the remaining monomeric lipids. If the nonpolymerizable lipids are PE's, then the photoinduced polymerization destabilizes the liposome with loss of aqueous contents. The permeability of the control dioleoylPC/SorbPC membranes was not affected by photopolymerization of SorbPC. The photodestabilization of dioleoylPE/SorbPC (3:1) liposomes required the presence of oligolamellar liposomes. NMR spectroscopy of extended bilayers of dioleoylPE/SorbPC (3:1) showed that the photopolymerization lowers the temperature for the appearance of 31P NMR signals due to the formation of isotropically symmetric lipid structures. These observations suggest the following model for the photoinduced destabilization of liposomes composed of PE/SorbPC: photopolymerization induced phase separation with the formation of enriched domains of PE, which allows the close approach of apposed regions of enriched PE lamellae and permits the formation of an isotropically symmetric structure between the lamellae. The formation of such an interlamellar attachment (ILA) between the lamellae of an oligolamellar liposome provides a permeability pathway for the light-stimulated leakage of entrapped water-soluble reagents. © 1992 American Chemical Society.
• Altbach, M. I., Mattingly, M. A., Brown, M. F., & Gmitro, A. F. (1991). Magnetic resonance imaging of lipid deposits in human atheroma via a stimulated-echo diffusion-weighted technique. Magnetic Resonance in Medicine, 20(2), 319-326.
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  PMID: 1775058;Abstract: NMR images of subintimal lipid deposits within the vessel walls of atherosclerotic human aortas were obtained at 37 and 27°C at 4.7 T. A combination of a stimulated-echo and pulsed-field gradients was used for suppressing the mobile tissue water relative to the less mobile tissue lipids. At 27°C there was also a substantial reduction of the subintimal lipid signal intensity, which is consistent with the characteristic phase transition of cholesteryl esters in human atheroma. These results represent the first direct detection of lipid deposits in nonprotruding atherosclerotic lesions with NMR imaging.
• Barry, J. A., Trouard, T. P., Salmon, A., & Brown, M. F. (1991). Low Temperature 2H NMR Spectroscopy of Phospholipid Bilayers Containing Docosahexaenoyl (22:6ω3) Chains. Biochemistry, 30, 8386-8394.
• Brown, M. F., & Rajamoorthi, K. (1991). Bilayers of Arachidonic Acid Containing Phospholipids Studied By 2H and 31P NMR Spectroscopy. Biochemistry, 30, 4204-4212.
• Gibson, N. J., & Brown, M. F. (1991). Membrane lipid influences on the energetics of the metarhodopsin I and metarhodopsin II conformational states of rhodopsin probed by flash photolysis.. Photochemistry and Photobiology, 54(6), 985-992.
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  PMID: 1775536;Abstract: We have investigated the relationship between rhodopsin photochemical function and the retinal rod outer segment (ROS) disk membrane lipid composition using flash photolysis techniques. Bovine rhodopsin was combined with various phospholipids to form recombinant membrane vesicles, in which the lipid acyl chain composition was maintained at that of egg phosphatidylcholine (PC), while the nature of the headgroups was varied. The ratio of metarhodopsin II (MII)/metarhodopsin I (MI) in these recombinants produced by an actinic flash was investigated as a function of pH, and compared with the photochemical activity observed for rhodopsin in native ROS membranes and dimyristoylphosphatidylcholine recombinants. In recombinants made with lipids derived from egg PC, as well as in native ROS membranes, MI and MII were found to be present in a pH-dependent, acid-base equilibrium on the millisecond timescale. The recombinants made with phospholipids containing unsaturated acyl chains were capable of full native-like MII production, but each demonstrated a titration curve with a different pK. In addition, some of the recombinants exhibited apparent deviations from the Henderson-Hasselbalch curve shape. The presence of either phosphatidylethanolamine (PE) or phosphatidylserine (PS) headgroups appeared to increase the amount of MII produced. This may result from alteration of the curvature free energy, in the case of PE, and from the influence of the membrane surface potential in the case of PS. An investigation of the effects of temperature on the MI-MII transition in native ROS membranes and the recombinants was also carried out.(ABSTRACT TRUNCATED AT 250 WORDS)
• Gibson, N. J., & Brown, M. F. (1991). Role of phosphatidylserine in the MI-MIII equilibrium of rhodopsin. Biochemical and Biophysical Research Communications, 176(2), 915-921.
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  PMID: 2025300;Abstract: Bovine rhodopsin was recombined with various phospholipids in which the lipid acyl chain composition was held constant at that of egg phosphatidylcholine (PC), while the identity of the headgroups was varied. The ratio of MII / MI produced in the recombinant membrane vesicles by an actinic flash was studied as a function of pH, and compared to the photochemical activity observed for rhodopsin in native ROS membranes. MI and MII were found to coexist in a pH-dependent, acid-base equilibrium on the millisecond timescale. Recombinants made with phospholipids containing unsaturated acyl chains were capable of full native-like MII production, but demonstrated titration curves with different pK values. The presence of phosphoethanolamine or phosphoserine headgroups increased the amount of MII produced. In the case of phosphatidylserine this may result from alteration of the membrane surface potential, leading to an increase in the local H+ activity. The results indicate that the Gibbs free energies of the MI and MII conformational states are influenced by the membrane bilayer environment, suggesting a possible role of lipids in visual excitation. © 1991 Academic Press, Inc.
• Thurmond, R. L., Dodd, S. W., & Brown, M. F. (1991). Molecular areas of phospholipids as determined by 2H NMR spectroscopy. Comparison of phosphatidylethanolamines and phosphatidylcholines. Biophysical Journal, 59(1), 108-113.
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  PMID: 2015377;PMCID: PMC1281123;Abstract: The role of lipid diversity in biomembranes is one of the major unsolved problems in biochemistry. One parameter of possible importance is the mean cross-sectional area occupied per lipid molecule, which may be related to formation of nonbilayer structures and membrane protein function. We have used 2H NMR spectroscopy to compare the properties of 1,2-diperdeuteriopalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE-d62) and 1,2-diperdeuteriopalmitoyl-sn-glycero-3-phosphocholine (DPPC-d62) in the L(α) phase. We find that DPPE has greater segmental order than DPPC, and that this increase in order is related to the smaller area per acyl chain found for DPPE. Values of the mean cross-sectional chain area are calculated using a simple diamond lattice model for the acyl chain configurational statistics, together with dilatometry data. The results obtained for the mean area per molecule are comparable with those from low angle x-ray diffraction studies.
• Thurmond, R. L., Lindblom, G., & Brown, M. F. (1991). Effect of bile salts on monolayer curvature of a phosphatidylethanolamine/water model membrane system. Biophysical Journal, 60(3), 728-732.
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  PMID: 1932556;PMCID: PMC1260117;Abstract: A partial phase diagram of the ternary system dioleoylphosphatidylethanolamine (DOPE)/sodium cholate/water has been determined using 31P Nuclear Magnetic Resonance (NMR) spectroscopy. In the absence of cholate, it is well known that the DOPE/water system forms a reversed hexagonal (H(II)) phase. We have found that addition of even small amounts of cholate to the DOPE/water system leads to a transition to a lamellar (L(α)) phase. At higher cholate concentrations, a cubic (I) phase (low water content) or a micellar solution (L1) phase (high water content) is present. Thus, cholate molecules have a strong tendency to alter the lipid monolayer curvature. Increasing the concentration of cholate changes the curvature of DOPE from negative (H(II) phase), through zero (L(α) phase), and finally to a phase of positive curvature (micellar solution). This observation can be rationalized in terms of the molecular structure of cholate, which is amphipathic and has one hydrophobic and one hydrophilic side of the steroid ring system. The cholate molecules have a tendency to lie flat on the lipid aggregate surface, thereby increasing the effective interfacial area of the polar head groups, and altering the curvature free energy of the system.
• Brown, M. F., & Söderman, O. (1990). Orientational anisotropy of nuclear spin relaxation in phospholipid membranes. Chemical Physics Letters, 167(1-2), 158-164.
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  Abstract: The observation that the spin-lattice relaxation (R1Z) rates of pure phospholipid lamellar phases depend only weakly on their orientation in the liquid-crystalline state is explained. A relaxation model in which either segmental or molecular motions are described by anisotropic rotational diffusion in an ordering potential (M.F. Brown, J. Chem. Phys. 77 (1982) 1576) can account for the available 2H R1Z data to within experimental error. One possibility is that rotational isomerization breaks the symmetry of the static electric field gradient, leading to an asymmetric residual tensor which is further modulated by molecular motions. © 1990.
• Brown, M. F., Salmon, A., Henriksson, U., & Söderman, O. (1990). Frequency Dependent 2H N.M.R. Relaxation Rates of Small Unilamellar Vesicles. Molecular Physics, 69, 379-383.
• Gibson, N. J., & Brown, M. F. (1990). Influence of pH on the MI-MII equilibrium of rhodopsin in recombinant membranes. Biochemical and Biophysical Research Communications, 169(3), 1028-1034.
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  PMID: 2363712;Abstract: Rhodopsin in native rod membranes and incorporated into egg phosphatidylcholine (egg PC) vesicles was studied at pH 5 and 7 at 28°C. Rhodopsin function, as monitored by the formation of metarhodopsin II (MII) from metarhodopsin I (MI) following an actinic flash, was found to be largely blocked in egg PC vesicles at pH 7. When the pH was lowered to 5, however, rhodopsin showed essentially equal activity in both native and egg PC membranes. This activity exceeded that found for rhodopsin in native membranes at pH 7. Phospholipid composition is thus shown to directly affect the MI ⇌ MII equilibrium, which in turn is linked to visual function. © 1990.
• Jansson, M., Thurmond, R. L., Trouard, T. P., & Brown, M. F. (1990). Magnetic Alignment and Orientational Order of Dipalmitoylphosphatidylcholine Bilayers Containing Palmitoyllyso-phosphatidylcholine. Chemistry and Physics of Lipids, 54, 157-170.
• Thurmond, R. L., Lindblom, G., & Brown, M. F. (1990). Influences of membrane curvature in lipid hexagonal phases studied by deuterium NMR spectroscopy. Biochemical and Biophysical Research Communications, 173(3), 1231-1238.
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  PMID: 2268326;Abstract: The presence of reversed hexagonal phase, HII, favoring lipids in membranes has been proposed to be significant in various biological processes. Therefore an understanding of the HII phase and the transition from the lamellar to hexagonal phase is of importance. We have applied deuterium NMR spectroscopy to study the bilayer and reversed hexagonal phases of 1-perdeuteriopalmitoyl-2-linoleoyl-sn-glycero-3-phosphoethanolamine. The difference in packing between the HII and Lα phases leads to smaller segmental order parameters in the former case. Since the order profiles are sensitive to the geometry of the aggregates, they can be used to extract structural information about the phases. We present a new means of calculating the radius of curvature, R1, for the HII phase from 2H NMR data. This method gives a value of R1 = 18.1 Å, which is in agreement with current understanding of the structure of the HII phase and with x-ray diffraction data. © 1990 Academic Press, Inc.
• Merickel, M. B., Carman, C. S., Brookeman, J. R., III, J. M., Brown, M. F., & Ayers, C. R. (1988). Identification and 3-D quantification of atherosclerosis using magnetic resonance imaging. Computers in Biology and Medicine, 18(2), 89-102.
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  PMID: 3356147;Abstract: Cardiovascular disease due to atherosclerosis is a leading cause of death in the United States as well as other developed countries. This paper describes the development of image processing, pattern recognition, and graphical display techniques to non-invasively quantify the atherosclerotic disease process using magnetic resonance imaging (MRI). We have demonstrated the ability to identify the soft tissue classes of (1) normal, smooth muscle wall, (2) fatty plaque, (3) complex, fibrous plaque, and (4) calcified plaque. The objective of this work hs been to combine functional information, such as plque tissue type, with structural information, represented by 3-D display of vessel structure, into a single composite display. The results of this work provide a 'high information content' display which will aid in the diagnosis and analysis of the atherosclerotic disease process, and permit detailed and quantitative studies to assess the effectiveness of therapies (e. g. changes in diet, exercise and drug administration).
• Pearlman, J. D., Zajicek, J., Merickel, M. B., Carman, C. S., Ayers, C. R., Brookeman, J. R., & Brown, M. F. (1988). High-resolution 1H NMR spectral signature from human atheroma. Magnetic Resonance in Medicine, 7(3), 262-279.
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  PMID: 3205143;Abstract: Coronary artery disease due to atherosclerosis takes the lives of approximately 550,000 Americans each year - an enormous toll. Put in economic terms, the cost to the United States alone has been estimated to exceed 60 billion dollars annually. We have found that well-resolved proton (1H) NMR spectra can be obtained from human atheroma (fatty plaque), despite its macroscopic solid appearance. The fraction of the total spectral intensity corresponding to the sharp 1H NMR signals is temperature dependent and approaches unity at body temperature (37°C). Studies of the total lipids extracted from atheroma and cholesteryl esters were conducted to identify the chemical and physical origin of the spectral signature. The samples were characterized through assignment of their chemical shifts and by measurement of their T1 and T2* relaxation times as a function of magnetic field strength. The results suggest that the relatively sharp 1H NMR signals from human atheroma (excluding water) are due to a mixture of cholesteryl esters, whose liquid-crystalline to isotropic fluid phase transition is near body temperature. Preliminary applications to NMR imaging of human atheroma are reported, which demonstrate early fatty plaque formation within the wall of the aorta. These findings offer a basis for noninvasive imaging by NMR to monitor early and potentially reversible stages of human atherogenesis.
• Wiedmann, T. S., Pates, R. D., Beach, J. M., Salmon, A., & Brown, M. F. (1988). Lipid-protein interactions mediate the photochemical function of rhodopsin. Biochemistry, 27(17), 6469-6474.
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  PMID: 3219348;Abstract: We have investigated the molecular features of recombinant membranes that are necessary for the photochemical function of rhodopsin. The magnitude of the metarhodopsin I to metarhodopsin II phototransient following a 25% ± 3% bleaching flash was used as a criterion of photochemical activity at 28°C and pH 7.0. Nativelike activity of rhodopsin can be reconstituted with an extract of total lipids from rod outer segment membranes, demonstrating that the protein is minimally perturbed by the reconstitution protocol. Rhodopsin photochemical activity is enhanced by phosphatidylethanolamine head groups and docosahexaenoyl (22:6ω3) acyl chains. An equimolar mixture of phosphatidylethanolamine and phosphatidylcholine containing 50 mol % docosahexaenoyl chains results in optimal photochemical function. These results suggest the importance of both the head-group and acyl chain composition of the rod outer segment lipids in the visual process. The extracted rod lipids and those lipid mixtures favoring the conformational change from metarhodopsin I to II can undergo lamellar (Lα) to inverted hexagonal (HII) phase transitions near physiological temperature. Interaction of rhodopsin with membrane lipids close to a Lα to HII (or cubic) phase boundary may thus lead to properties which influence the energetics of conformational states of the protein linked to visual function. © 1988 American Chemical Society.
• Salmon, A., Dodd, S. W., Williams, G. D., Beach, J. M., & Brown, M. F. (1987). Configurational statistics of acyl chains in polyunsaturated lipid bilayers from 2H NMR. Journal of the American Chemical Society, 109(9), 2600-2609.
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  Abstract: To gain a better understanding of the biological roles of polyunsaturated phospholipids, deuterium (2H) NMR studies have been conducted of 1-perdeuteriopalmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine, an asymmetric or mixed-chain saturated-polyunsaturated phospholipid, in the liquid crystalline (Lα) phase. The palmitoyl (16:0) chain at the glycerol sn-1 position was labeled with 2H by perdeuteration, whereas the polyunsaturated, docosahexaenoyl (22:6ω3) chain at the sn-2 position was unlabeled, i.e., protiated. The 2H NMR results were compared to studies of 1,2-diperdeuteriopalmitoyl-sn-glycero-3-phosphocholine, in which both the sn-1 and sn-2 palmitoyl chains were perdeuterated, as well as 1-palmitoyl-2-perdeuteriopalmitoyl-sn-glycero-3-phosphocholine, in which only the sn-2 chain was perdeuterated. Multilamellar phospholipid dispersions containing 50 wt % H2O were employed, and 2H NMR spectra were obtained using quadrupolar echo methods at a magnetic field strength of 8.5 T. The experimental 2H NMR spectra were numerically deconvolved (de-Paked) to yield subspectra corresponding to the parallel bilayer orientation with respect to the main applied magnetic field. The increased resolution of the de-Paked subspectra enabled profiles of the segmental order parameters of the individual C-2H bonds, denoted by |SCD(i)|, to be derived as a function of chain position. Significant differences in the 2H NMR spectra and derived |SCD(i)| profiles of the per-2H-16:0 chains of the polyunsaturated and saturated bilayers were found. Based on simplified statistical mechanical theories, the differences can be interpreted in terms of an increase in the configurational freedom of the palmitoyl chains in the polyunsaturated bilayer, relative to bilayers of phosphatidylcholines with two identical saturated chains. The increased configurational freedom may correspond to an increase in the equilibrium area per chain in the case of the polyunsaturated bilayer. Possible further interpretations of the results in terms of the thickness of the hydrocarbon region and the presence or lack of interdigitation of the polyunsaturated and saturated acyl chains are also briefly discussed. We conclude that the configurational properties of the acyl chains of polyunsaturated bilayers are significantly different from those of saturated phospholipid bilayers. © 1987 American Chemical Society.
• Zajicek, J., Pearlman, J. D., Merickel, M. B., Ayers, C. R., Brookeman, J. R., & Brown, M. F. (1987). High-resolution proton NMR spectra of human arterial plaque. Biochemical and Biophysical Research Communications, 149(2), 437-442.
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  PMID: 3426583;Abstract: Well-resolved proton (1H) NMR spectra of solid human arterial plaque can be acquired. Studies have been carried out of human fatty plaque obtained postmortem (ex vivo), the total lipids extracted from human atheroma, and a model mixture of cholesteryl esters whose lipid composition resembles that of human atheroma. In each case, well-resolved 1H NMR spectra were obtained at body temperature (37°C), with little or no underlying broad signal. Such sharp 1H NMR spectra are typical of isotropic fluids, whereas solid and liquid-crystalline materials give rise to much broader spectral lines. The results suggest the sharp 1H NMR spectra of human atheromatous lesions at body temperature are due largely to the presence of intracellular and extracellular droplets of cholesteryl esters in the isotropic liquid phase. These findings provide a necessary basis for use of 1H NMR techniques to image quantitatively the lipid constituents of human atheroma in vivo, and to study their chemical and physical properties. © 1987.
• Ellena, J. F., Pates, R. D., & Brown, M. F. (1986). 31P NMR spectra of Rod Outer Segment and Sarcoplasmic Reticulum membranes show no evidence of immobilized components due to lipid-protein interactions. Biochemistry, 25(13), 3742-3748.
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  PMID: 3741833;Abstract: 31P NMR studies of rod outer segment (ROS) and sarcoplasmic reticulum (SR) membranes have been performed under conditions where broad and narrow spectral components can be clearly resolved. Control studies of an anhydrous, solid powder of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), as well as aqueous binary mixtures of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), demonstrate clearly that broad spectral components can be detected. For the codispersions of DSPC and DOPC in the mixed-phase region at 22°C, the 31P NMR spectra consist of a superposition of a broad component and a narrow, axially symmetric component, due to coexisting solid and liquid-crystalline domains, which are in slow exchange on the 31P NMR time scale. The 31P NMR spectra of the native ROS and SR membranes, however, consist of only a narrow component, to within experimental error, indicating that most or all of the phospholipids are in the liquid-crystalline (Lα) phase at 22°C. The above conclusions are in agreement with many, but not all, previous studies [see, e.g., Yeagle, P. L. (1982) Biophys. J. 37, 227-239]. It is estimated that at most 10% of the phospholipids in the ROS and SR membranes could give rise to broad 31P NMR spectral components, similar to those seen for anhydrous or solid-phase lipids, corresponding to ∼7 phospholipids/rhodopsin molecule and ∼11 phospholipids/ Ca2+-ATPase molecule, respectively. © 1986 American Chemical Society.
• Ellena, J. F., Pates, R. D., Brown, M. F., Selinsky, B. S., & Yeagle, P. L. (1986). Erratum: 31P NMR spectra of rod outer segment and sarcoplasmic reticulum membranes show no evidence of immobilized components due to lipid-protein interactions (Biochemistry (1986) 25:13 (3742-3748)). Biochemistry, 25(26), 8473-.
• Brown, M. F., & Williams, G. D. (1985). Membrane NMR: a dynamic research area. Journal of Biochemical and Biophysical Methods, 11(2-3), 71-81.
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  PMID: 3897353;Abstract: Recent NMR relaxation studies of lipid bilayers and biomembranes are explained and briefly discussed. The results of both 2H and 13C NMR investigations suggest that, in addition to rapid local fluctuations of the hydrocarbon chains, slower, more collective motions of the bilayer exist. When the influence of the latter is recognized and properly accounted for, the contribution from local motions can be used to estimate a value for the microviscosity of the bilayer which corresponds to that of a simple n-paraffinic liquid. In general, the dynamic behavior of lipid bilayers as studied by NMR appears quite similar to that of simpler liquid crystals. © 1985.
• Brown, M. F., Ellena, J. F., Trindle, C., & Williams, G. D. (1985). Frequency dependence of spin-lattice relaxation times of lipid bilayers. The Journal of Chemical Physics, 84(1), 465-470.
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  Abstract: 2H and 13C spin-lattice (T1) relaxation time studies of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in the lamellar, liquid crystalline (Lα) phase are discussed. It is shown that the T1-1 results as a function of Larmor frequency ω0 are statistically better described by an ω0-1/2 dependence than by an ω 0-1 or ω0-2 dependence. © 1985 American Institute of Physics.
• Miljanich, G. P., Brown, M. F., Mabrey-Gaud, S., Dratz, E. A., & Sturtevant, J. M. (1985). Thermotropic behavior of retinal rod membranes and dispersions of extracted phospholipids. The Journal of Membrane Biology, 85(1), 79-86.
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  PMID: 4020856;Abstract: High sensitivity, differential scanning calorimetry studies of vovine retinal rod outer segment (ROS) disk membranes and aqueous dispersions of the extracted ROS phospholipids have been performed. ROS disk membranes were found to exhibit a broad peak of excess heat capacity with a maximum at less than about 3°C, ascribable to a gel-to-liquid crystalline phase transition of traction of the phospholipids. A similar thermotropic transition was observed for aqueous dispersions of the total extracted and purified ROS phospholipids. Comparison of the results obtained for the dispersion of total ROS phospholipids to those of the purified head group fractions. suggests that the thermotropic behavior reffects a gel-to-liquid crystalline transition, leading to lateral phase separation, involving those phosphatidylcholine (PC) molecules containing saturated fatty acylchains, possibley together with the highest melting ROS phosphatidylethanolamine (PE) and phosphatidylserine (PS) components. The interpretation of the thermal behavior of the ROS disk membranes depends on whether the transition is assumed to derive from the ROS PC and/or PE/PS fractions, and whether the transbilayer arrangement of the ROS phospholipids is assumed to be symmetric or asymmetric. The calorimetric data can be simply explained in terms of an asymmetric distribution of the major ROS disk membrane phospholipids (G.P. Miljanich et al., J. Membrane Biol.60:249-255, 1981). In this case, the transition would arise from the PE/PS fractions in the outer ROS disk membrane monolyer, and the anticipated transition from the PC in the inner monolayer would be broadened due to interaction with cholesterol. For the ROS membranes at higher temperatures, two additional, irreversible transitions are observed at 57 and 72°C, corresponding to the thermal denauturation of opsin and rhodopsin, respectively. © 1985 Springer-Verlag.
• Williams, G. D., Beach, J. M., Dodd, S. W., & Brown, M. F. (1985). Dependence of deuterium spin-lattice relaxation rates of multilamellar phospholipid dispersions on orientational order. Journal of the American Chemical Society, 107(24), 6868-6873.
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  Abstract: 2H NMR studies of a homologous series of 1,2-diacyl-sn-glycero-3-phosphocholines with perdeuterated saturated chains, ranging in length from C12:0 to C16:0, have been performed with use of quadrupolar echo techniques at a resonance frequency of 55.4 MHz. Randomly oriented, multilamellar dispersions containing 50 wt % H2O in the liquid crystalline (Lα) phase have been employed. The 2H spin-lattice relaxation times (T1) and C-2H bond segmental order parameters (SCD) of each of the resolved quadrupolar splittings have been obtained from the powder-type spectra, corresponding to a random distribution of orientations, as well as from the 0°C oriented subspectra obtained by numerical deconvolution (de-Pakeing). Evidence that the spin-lattice relaxation rate profiles as a function of chain position T1-1(i) are related to the corresponding order profiles SCD(i) by a square-law functional dependence has been obtained, indicative of a contribution from relatively slow fluctuations in the local bilayer ordering to the relaxation. The results suggest that two broad classes of motions influence the 2H spin-lattice relaxation rates of lipid bilayers: rapid local motions, most likely due to bond rotational isomerizations and long-axis rotational diffusion of the lipid chains, as well as slower director fluctuations as found in other liquid crystalline mesophases. © 1985 American Chemical Society.
• Siminovitch, D. J., Brown, M. F., & Jeffrey, K. R. (1984). 14N NMR of lipid bilayers: Effects of ions and anesthetics. Biochemistry, 23(11), 2412-2420.
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  PMID: 6477874;Abstract: The interaction of divalent and trivalent metal cations, ferricyanide, a lipophilic ion (tetraphenylborate), and a local anesthetic (tetracaine) with the phosphocholine head group of egg lecithin was investigated by using wide-line 14N and 31P NMR. Measurements of the 14N quadrupolar splittings in the presence of a variety of perturbing agents demonstrated that the 14N NMR technique can be used to directly monitor ion or anesthetic binding. The 14N quadrupolar splitting (ΔvQ) is a measure of the order parameter of the Cβ-N bond segment, and changes in ΔvQ as large as 3.5 kHz were observed. Moreover, a comparison of the changes in the quadrupolar splittings induced by the binding of ions or anesthetics provided a sensitive method of discriminating between these perturbing agents in their ability to alter the orientational order of the Cβ-N bond segment of the phosphocholine moiety. Without exception, addition of metal ions or anesthetics always resulted in a decrease of the 14N ΔvQ. This reduction reflects a change in the average orientation or degree of motional averaging at the Cβ-N bond segment position. In the case of metal ion binding, the strength of the interaction increased with the charge of the metal ion in the order Ca2+ < Ln3+, in agreement with a previous 2H NMR study [Akutsu, H., & Seelig, J. (1981) Biochemistry 20, 7366-7373]. However, distinct differences were also noted between ions of the same charge, and in the case of the trivalent lanthanide ions, the 14N ΔvQ decreased in the sequence La3+ > Pr3+ > Eu3+ > Lu3+, following the order of the lanthanide contraction. The 14N and 31P line shapes in the presence of lanthanide ions showed that it is possible to clearly distinguish between the effects of paramagnetic (Pr3+, Eu3+, Dy3+, Tm3+) and diamagnetic (La3+, Lu3+) ions. Unusual, distinctive "asymmetric" 14N NMR line shapes were observed in the presence of the paramagnetic lanthanides, apparently due to incomplete averaging of the magnetic dipolar interaction between the bound lanthanide ion and the nitrogen nucleus. Addition of the lipophilic anion tetraphenylborate led to a slight reduction in the 14N ΔvQ, together with a dramatic decrease in the absolute value of the 31P chemical shielding anisotropy (Δσ). By contrast, the reduction of the 14N ΔvQ due to the presence of tetracaine was accompanied by a substantial increase in the 31P |Δσ|. In general, the measurements of the 14N ΔvQ as well as the 14N spin-lattice (T1) relaxation times support the notion that there is a rapid exchange of ions from the bulk medium to the ligand binding sites and among the various binding sites, in agreement with 2H NMR studies. The above results suggest that 14N NMR can provide a useful complement to 31P and 2H NMR techniques in studies of the influence of various perturbing agents on the orientational order and dynamics of the phosphocholine head groups in membranes. © 1984 American Chemical Society.
• Siminovitch, D. J., Rance, M., Jeffrey, K. R., & Brown, M. F. (1984). The quadrupolar spectrum of a spin I = 1 in a lipid bilayer in the presence of paramagnetic ions. Journal of Magnetic Resonance, 58(1), 62-75.
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  Abstract: The NMR signal from selectively deuterated molecules in a lipid bilayer where there are paramagnetic ions present in the aqueous region is influenced by both the nuclear quadrupole interaction and the dipolar interaction between the deuterium nuclei and the surrounding ions. The quadrupolar split powder pattern is no longer symmetric about the center of the spectrum. The spectra which result from the use of the quadrupole echo pulse sequence are quite complex since this sequence does not refocus the dephasing due to the dipolar interaction. A new pulse sequence which refocuses both the dipolar and quadrupolar interactions is suggested. Both 2H and 14N spectra from lipid molecules with the phosphatidylcholine headgroup obtained with the conventional quadrupole echo sequence and the new sequence are compared with predictions of density matrix theory calculations. There is excellent agreement between the experimental and simulated spectra. © 1984.
• Brown, M. F. (1983). Theory of spin-lattice relaxation in lipid bilayers and biological membranes. Dipolar relaxation. The Journal of Chemical Physics, 80(6), 2808-2831.
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  Abstract: In this work we have calculated spin-lattice (T1) relaxation time expressions for the homo- and heteronuclear dipolar relaxation of lipid bilayers, in addition to the heteronuclear Overhauser enhancement (NOE), using correlation functions derived previously. The results can be applied to the analysis of the 1H and 13C T1 times of lipid bilayers and the 13C-1H NOE. Three different models for the segmental fluctuations of the membraneous lipid molecules have been considered: (i) a simple diffusion-type model for the local segmental motions; (ii) a noncollective model in which relatively slow bilayer fluctuations are described by a single correlation time; and (iii) a collective model for the slow motions characterized by a continuous distribution of correlation times. For the diffusion model, the dependence on the bilayer orientation, order parameters 〈P2〉 and 〈P4〉, and the diffusion tensor anisotropy have been included in a general manner. Depending on the degree of segmental ordering and the anisotropy of the diffusion tensor, the maximum 13C-1H NOE can be either greater or less than the value of 2.988 obtained in the absence of an ordering potential. The various relaxation models were fit to 13C T1 data recently obtained for vesicles of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) at seven different magnetic field strengths, i.e., resonance frequencies, in the liquid crystalline state. A simple diffusion-type model (i) based on analogies to paraflinic liquids provides a very poor fit to the above 13C T1 data as a function of temperature and frequency, even for extreme values of the ordering and diffusion tensor anisotropy,and thus can be rejected at the present time. The 13C results can be fit satisfactorily over the range 15.0-126 MHz by models which include contributions from relatively slow bilayer fluctuations. A noncollective model (ii) with three or four adjustable parameters or a collective model (iii) with two parameters both describe the data to within experimental error. At present, the 13C T1 results suggest that the relaxation of the bilayer hydrocarbon region, in the liquid crystalline state, can best be accounted for by a collective model with a relaxation expression of the type T1-1≅Aτf(2)+BCH2ω1 as concluded from a similar analysis of the 2H T1 data for DPPC multilamellar dispersions. In the above expression, τf(2) is the correlation time for the local motions, SCH(=SCD) is the observed bond segmental order parameter, ω1 is the resonance frequency, and A and B are constants which depend on the nucleus considered. Thus, the observed relaxation rate includes contributions from fast or local-type motions, in addition to cooperative fluctuations of a more long-range character. For the collective model, extrapolation of the 13C T1-1 values obtained for the DPPC vesicles to infinite frequency yields estimates of τf(2) which agree with those calculated from the frequency-independent T1-1 rates of n-hexadecane at the same absolute temperature, suggesting that the segmental microviscosities of the two systems are similar, in agreement with 2H NMR studies. © 1984 American Institute of Physics.
• Brown, M. F. (1983). Unified picture for spin-lattice relaxation of lipid bilayers and biomembranes. The Journal of Chemical Physics, 80(6), 2832-2836.
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  Abstract: The present study compares and interprets the 1H, 2H, and 13C spin-lattice (T1) relaxation times of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), in the liquid crystalline phase, in terms of models for the molecular dynamics of lipid bilayers. The 1H T1 times of the DPPC bilayer hydrocarbon region at two frequencies and 13C T1 data at seven frequencies, for which the relaxation is dipolar in origin, as well as the 2H T 1 data at three frequencies, due to the quadrupolar interaction, can be unified and interpreted in terms of a collective model for order fluctuations. In normalizing the 13C T1 data to the 1H and 2H T1 values, a vibrationally corrected 13C-1H distance parameter of rCH0=1.14 Å has been assumed, rather than the equilibrium bond length of 1.09 Å. The analysis suggests that the behavior of the individual acyl chain segments of lipid bilayers, in the liquid crystalline phase, is similar to that of molecules in nematic fluids. © 1984 American Institute of Physics.
• Brown, M. F., Ribeiro, A. A., & Williams, G. D. (1983). New view of lipid bilayer dynamics from 2H and 13C NMR relaxation time measurements. Proceedings of the National Academy of Sciences of the United States of America, 80(14 I), 4325-4329.
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  PMID: 6576340;PMCID: PMC384030;Abstract: Natural abundance 13C spin-lattice (T1) relaxation time measurements are reported for unilamellar vesicles of 1,2-dipalmitoylphosphatidylcholine (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), in the liquid crystalline phase, at magnetic field strengths of 1.40, 1.87, 2.35, 4.23, 7.05, 8.45, and 11.7 tesla (resonance frequencies of 15.0, 20.0, 25.1, 45.3, 75.5, 90.5, and 126 MHz, respectively), and the results are compared to previous 2H T1 studies of multilamellar dispersions. For both the 13C and 2H T1 studies, a dramatic frequency dependence of the relaxation was observed. At superconducting magnetic field strengths (4.23-11.7 tesla), plots of the 13C T1-1 relaxation rates as a function of acyl chain segment position clearly reveal the characteristic 'plateau' signature of the liquid crystalline phase, as found previously from 2H NMR studies. The dependence of T1-1 on ordering, determined previously from 2H NMR, and the T1-1 dependence on frequency, determined from both 13C and 2H NMR studies, suggest that a unified picture of the bilayer molecular dynamics can be provided by a simple relaxation law of the form T1-1 ≃ Aτf + BS2 (C-H) ω0(- 1/2 ). In the above expression, A and B are constants, S(C-H)(=S(C-D) is the bond segmental order parameter, and ω0 is the nuclear Larmor frequency. The first (A) term includes contributions from fast, local segmental motions characterized by the effective correlation time τf, whereas the second (B) term describes slower, collective fluctuations in the local ordering. The value of τf ≃ 10-11 sec, obtained by extrapolating T1-1 to infinite frequency, suggests that the segmental microviscosity of the bilayer hydrocarbon region does not differ appreciably from that of the equivalent n-paraffinic liquids of similar chain length.
• Sefcik, M. D., Schaefer, J., Stejskal, E. O., McKay, R. A., Ellena, J. F., Dodd, S. W., & Brown, M. F. (1983). Lipid bilayer dynamics and rhodopsin-lipid interactions: New approach using high-resolution solid-state 13C NMR. Biochemical and Biophysical Research Communications, 114(3), 1048-1055.
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  PMID: 6615501;Abstract: High-resolution, solid-state 13C NMR spectra have been obtained for unsonicated multilamellar dispersions of 1,2-dilauryl-sn-glycero-3-phosphocholine (DLPC), recombinant membranes containing DLPC and rhodopsin, and native retinal rod disk membranes. The roles of 1H dipolar decoupling, 1H-13C cross-polarization, and magic-angle sample spinning have been investigated. Rotating-frame 13C relaxation times have been measured and are discussed in terms of lipid bilayer dynamics and rhodopsin-lipid interactions. © 1983.
• Brown, M. F. (1982). Theory of spin-lattice relaxation in lipid bilayers and biological membranes. 2H and 14N quadrupolar relaxation. The Journal of Chemical Physics, 77(3), 1576-1599.
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  Abstract: Based on a previous, more approximate treatment [M. F. Brown, J. Magn. Reson. 35, 203 (1979)], expressions are derived for the quadrupolar spin-lattice (T1) relaxation rates of 2H and 14N in lipid bilayers. Results are presented for the most general, anisotropic rotational diffusion model describing the segmental or molecular reorientation in lipid bilayers, and the analysis is extended to include relatively slow fluctuations of the local director with respect to the macroscopic bilayer normal. Numerically computed values of T1 for the diffusion model suggest that, even for extremes of ordering and motional anisotropy, such a model cannot by itself quantitatively account for the observed 2H T1 values of multilamellar dispersions of 1,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC), in the liquid crystalline state, as a function of temperature and frequency. The contribution from relatively low frequency motions is modeled in terms either (i) a simple noncollective model in which the slow motions are described in terms of a single effective correlation time, or (ii) a collective model in which the relatively slow reorientation is described by a distribution of correlation times, corresponding to collective fluctuations of the instantaneous director. The experimentally observed dependence of the 2H T1 relaxation rates on the acyl chain segmental order parameter SCD and the resonance frequency ω0 are most consistent with a collective model for slow molecular reorientations in lipid bilayers. The 2H T1 data for the saturated DPPC bilayer, in the liquid crystalline state, can be quantitatively described by a relaxation law of the form T1-1 = Aτf + BSCD2 ω0-1/2 as observed for simpler nematic and smectic liquid crystals. The first (A) term is suggested to correspond primarily to trans-gauche isomerizations of the lipid acyl chains, while the (B) term describes collective bilayer modes which predominantly influence the frequency dependence of the relaxation. In contrast to earlier conclusions [M. F. Brown et al., J. Chem. Phys. 70, 5045 (1979)], the dominant contribution to the 2H T1 relaxation rates of the saturated DPPC bilayer may arise from collective order fluctuations rather than fast local motions. The value of τf∼10-11 s obtained by extrapolating T1-1 to infinite frequency or zero ordering is consistent with the correlation times calculated from 2H or 13C T1 data for n-alkanes of equivalent chain lengths, suggesting that the microviscosity of the bilayer hydrocarbon region is not appreciably different from that of paraffinic liquids. © 1982 American Institute of Physics.
• Brown, M. F., Deese, A. J., & Dratz, E. A. (1982). Proton, carbon-13, and phosphorus-31 NMR methods for the investigation of rhodopsin-lipid interactions in retinal rod outer segment membranes. Methods in Enzymology, 81(C), 709-728.
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  PMID: 7098912;
• Brown, M. F., & Davis, J. H. (1981). Orientation and frequency dependence of the deuterium spin-lattice relaxation in multilamellar phospholipid dispersions: implications for dynamic models of membrane structure. Chemical Physics Letters, 79(3), 431-435.
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  Abstract: Rapid lateral diffusion of phospholipid molecules in multilamellar dispersions is shown to prevent the observation, in powder pattern spectra, of the orientation dependence of 2H nuclear magnetic spin-lattice relaxation. The spin-lattice relaxation rate is found to have a frequency dependence suggestive of collective director fluctuations. © 1981.
• Deese, A. J., Dratz, E. A., & Brown, M. F. (1981). Retinal rod outer segment lipids form bilayers in the presence and absence of rhodopsin: a 31P NMR study. FEBS Letters, 124(1), 93-99.
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  PMID: 7215559;
• Brown, M. F. (1979). Deuterium relaxation and molecular dynamics in lipid bilayers. Journal of Magnetic Resonance (1969), 35(2), 203-215.
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  Abstract: The quadrupolar relaxation of deuterium-labeled lipid bilayers has been analyzed using standard Redfield theory and is discussed with regard to the problem of chain segmental motion and order in membranes. Considering the segmental reorientation as a stochastic process, the T1 and T2 relaxation rates are interpreted in terms of the rate of motion, characterized by one or more correlation times τ2M, and statistical amplitude, characterized by the segmental order parameter SCD. For the case of phospholipid bilayers with |SCD| ≲ 0.2, the relaxation rates are predominantly determined by the rate of motion, rather than the ordering. Recently obtained T, relaxation data for selectively deuterated and perdeuterated multilamellar dispersions of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine are analyzed and compared to the results of previous carbon-13 T1 relaxation studies. The available experimental results suggest that the fast segmental motions affecting T1 in these systems can be treated to a reasonable degree of approximation in terms of a single effective correlation time. © 1979.
• Brown, M. F., Seelig, J., & Häberlen, U. (1979). Structural dynamics in phospholipid bilayers from deuterium spin-lattice relaxation time measurements. The Journal of Chemical Physics, 70(11), 5045-5053.
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  Abstract: The quadrupolar spin-lattice (T1) relaxation of deuterium labeled phospholipid bilayers has been investigated at a resonance frequency of 54.4 MHz. T1 measurements are reported for multilamellar dispersions, single bilayer vesicles, and chloroform/methanol solutions of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), selectively deuterated at ten different positions in each of the fatty acyl chains and at the sn-3 carbon of the glycerol backbone. At all segment positions investigated, the T 1 relaxation times of the multilamellar and vesicle samples of DPPC were found to be similar. The profiles of the spin-lattice relaxation rate (1/T1) as a function of the deuterated chain segment position resemble the previously determined order profiles [A. Seelig and J. Seelig, Biochem. 13, 4839 (1974)]. In particular, the relaxation rates are approximately constant over the first part of the fatty acyl chains (carbon segments C3-C9), then decreasing in the central region of the bilayer. In chloroform/methanol solution, by contrast, the relaxation rates decrease continuously from the glycerol backbone region to the chain terminal methyl groups. The contributions from molecular order and motion to the T1 relaxation rates have been evaluated and correlation time profiles derived as a function of chain position. The results suggest that the motions of the various methylene segments are correlated in the first part of the fatty acyl chains (C3-C9), occurring at frequencies up to 1/τc∼1010Hz. Beyond C9, the rate and amplitude of the chain segmental motions increase, approaching that of simple paraffinic liquids in the central region of the bilayer (1/τc≃1011Hz). The T1 relaxation rates of multilamellar dispersions of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) deuterated at the 9, 10 double bond of the sn-2 chain were also determined and found to be significantly faster than those of the CD2 chain segments of DPPC bilayers. This is most likely due to the larger size and correspondingly slower motion of the chain segment containing the double bond. At segments close to the lipid-water interface the rate of motion is considerably less than in the hydrocarbon region of the bilayer. © 1979 American Institute of Physics.
• Omar, S., Brown, M. F., Silver, P., & Schleich, T. (1979). Histidyl and tyrosyl residue ionization studies of subtilisin novo. Biochimica et Biophysica Acta, 578(2), 261-268.
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  PMID: 39621;Abstract: The low field portion of the 360 MHz 1H nuclear magnetic resonance spectrum of phenylmethanesulfonyl-subtilisin Novo (EC 3.4.21.14) has been studied as a function of pH. Analysis of the pH-induced chemical shift changes occurring between 6 to 7 ppm revealed five classes of ionizable residues with pK values (uncorrected) of 10.3, 10.7, 10.7, 10.8, and 11.0. With a single exception, the titration curves can be fit by assuming a simple proton ionization equilibrium. Four classes of low intensity broad resonances, assigned to the histidyl residues, are observed between 8 and 9 ppm. Uncorrected pK values of 5.4, 5.7, 6.0, and 6.4 were determined for the residues comprising each of these classes. The spectral data are consistent with protonation of one or more histidyl residues upon acid induced denaturation of the protein. These results are compared with those of analogues studies performed by the use of other techniques. © 1979.
• Brown, M. F., & Seelig, J. (1978). Influence of cholesterol on the polar region of phosphatidylcholine and phosphatidylethanolamine bilayers. Biochemistry, 17(2), 381-384.
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  PMID: 619997;Abstract: The structural changes in the polar head group region of unsonicated bilayer membranes of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine produced by addition of cholesterol have been determined using deuterium and phosphorus-31 NMR. Incorporation of up to 50 mol % cholesterol produces little change in the phosphorus-31 chemical shielding anisotropies, compared with the values in pure bilayers above the phase transition temperatures, while some of the deuterium quadrupole splittings are reduced by almost a factor of two. Adjustment of the head group torsion angles by only a few degrees accounts for the observed spectral changes. Addition of cholesterol therefore has opposite effects on the hydrocarbon and polar regions of membranes: although cholesterol makes the hydrocarbon region gel-like, with an increased probability of trans conformations, the conformation of the polar head groups is very similar to that found in the liquid crystalline phase of pure phospholipid bilayers. © 1978 American Chemical Society.
• Brown, M. F., & Schleich, T. (1977). Resolution of independently titrating spectral components of the ultraviolet circular dichroism of subtilisin enzymes by matrix rank analysis. Biochimica et Biophysica Acta, 485(1), 37-51.
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  PMID: 20969;Abstract: The ultraviolet circular dichroism of di-isopropylphophoryl-subtilisins Carlsberg and Novo (EC 3.4.21.14) has been examined as a function of pH. The CD of these enzymes below 260 nm is invariant over the pH interval 4 to 12, below or above which spectral changes occur suggesting a transition to a random coil form. Above pH 8 contributions due to the ionization of tyrosyl residues appear in the CD above 260 nm as bands shifted to longer wavelengths. Three independently titratable components, obtained by matrix rank analysis, account for the observed CD spectral changes above 260 nm of Dip-subtilisin Carlsberg in the pH interval 8 to 12. By contrast, two components were derived for the Novo enzyme. The identities of the matrix rank component were surmised from their apparent pK1 values. One component of both subtilisin enzymes corresponds to the CD of the "buried" or irreversibly titratable tyrosyl residues of the enzyme. The other matrix rank components correspond to the CD of the "exposed' or freely ionizable tyrosyl residues. These residues are optically active only in the ionized state. Two types of "expressed" tyrosyl residues, arising because of differing sensitivity to the ionization of the "partially buried" or abnormally titrating tyrosyl residues, are evident in Dip-subtilisin Carlsberg. A pH-induced local conformational change in this enzyme is proposed to account for this behavior. The "partially buried" tyrosyl residues of both subtilisins appear to be devoid of optical activity in either the tyrosyl or tyrosylate form. © 1977.
• Brown, M. F., & Seelig, J. (1977). Ion-induced changes in head group conformation of lecithin bilayers. Nature, 269(5630), 721-723.
• Brown, M. F., Miljanich, G. P., & Dratz, E. A. (1977). Interpretation of 100- and 360-MHz proton magnetic resonance spectra of retinal rod outer segment disk membranes. Biochemistry, 16(12), 2640-2648.
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  PMID: 889780;Abstract: Well resolved proton nuclear magnetic resonance (1H NMR) spectra of bovine retinal rod outer segment (ROS) disk membranes have been obtained at 100 and 360 MHz. The resolved 1H resonances of the ROS membranes are due to phospholipids, with little contribution from rhodopsin. The spectra of both the ROS membranes and bilayer vesicles prepared from purified ROS phospholipids (liposomes) appear to represent a superposition of relatively sharp resonance components and a broad, underlying background. The distribution between sharp and broad spectral components is sensitive to sonication and temperature. The percentage of choline methyl protons which are resolved in the ROS membrane spectra as sharp resonance components increases from approximately 35 to 100% and the average of the lipid hydrocarbon chain protons from approximately 20 to 40% over the temperature range 5-50°C. The motional state of terminal CH3 groups on the polyunsaturated docosahexenoic acid (C22:6ω3) side chains cannot be very different from those on the less unsaturated side chains, since the observed terminal CH3 resonance consists of components from the C22:6ω3 and other side chains in proportions which reflect their composition ratios. The observation of a comparable fraction of phospholipids yielding high-resolution spectral components and similar resonance line widths for the ROS membranes and ROS liposomes suggests that rhodopsin does not greatly alter the lower frequency segmental motions of phospholipids in the ROS membrane. The NMR data are discussed in terms of models for the organization of phospholipids in the disk membrane and their interaction with rhodopsin.
• Brown, M. F., Milljanich, G. P., & Dratz, E. A. (1977). Proton spin lattice relaxation of retinal rod outer segment membranes and liposomes of extracted phospholipids. Proceedings of the National Academy of Sciences of the United States of America, 74(5), 1978-1982.
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  PMID: 266718;PMCID: PMC431056;
• Brown, M. F., Omar, S., Raubach, R. A., & Schleich, T. (1977). Quenching of the tyrosyl and tryptophyl fluorescence of subtilisins Carlsberg and Novo by iodide. Biochemistry, 16(5), 987-992.
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  PMID: 843526;Abstract: The tyrosyl and tryptophyl fluorescence of diisopropylphosphorylsubtilisins Carlsberg and Novo, respectively, is quenched efficiently by I- but is not significantly affected by Cs+. The I- quenching data were analyzed using a modified Stern-Volmer treatment (Lehrer, S. S. (1971), Biochemistry 10, 3254), yielding values for the effective fraction of accessible protein fluorescence of 90-95 and 88-92% for the tyrosyl and tryptophyl emission of diisopropylphosphorylsubtilisins Carlsberg and Novo, respectively. Similar values were obtained at pH 5 and 7. The effective collisional quenching constant depends on pH in a manner suggesting the participation of protein surface charge in the quenching mechanism. Significant singlet energy transfer (efficiency = 0.52) from tyrosyl to tryptophyl residues was inferred from the excitation spectra of diisopropylphosphorylsubtilisin Novo. The very low efficiency of energy transfer to Trp-113 in diisopropylphosphorylsubtilisin Carlsberg suggests that Trp-105 and Trp-241 are the acceptors of tyrosyl emission in the homologous Novo enzyme. The unusually low quantum yield of Trp-113 in diisopropylphosphorylsubtilisin Carlsberg together with the tryptophyl fluorescence quenching behavior of the Novo enzyme suggests that this residue is "buried" and inaccessible to quenching in both enzymes. The tyrosyl quenching behavior of diisopropylphosphorylsubtilisin Carlsberg is consistent with the high degree of solvent exposure of aromatic residues evident in the x-ray model of subtilisin Novo.
• Brown, M. F., Miljanich, G. P., Franklin, L. K., & Dratz, E. A. (1976). 1H-NMR studies of protein-lipid interactions in retinal rod outer segment disc membranes. FEBS Letters, 70(1-2), 56-60.
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  PMID: 992076;
• Brown, M. F., & Schleich, T. (1975). Circular dichroism and gel filtration behavior of subtilisin enzymes in concentrated solutions of guanidine hydrochloride. Biochemistry, 14(14), 3069-3074.
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  PMID: 238582;Abstract: The circular dichroism of diisopropylphosphorylsubtilisins Novo and Carlsberg in both the near- and farultraviolet spectral regions is unaltered by concentrations of guanidine hydrochloride as high as 4 M at neutral pH. At concentrations of guanidine hydrochloride greater than 4 M slow irreversible time-dependent changes, apparently obeying second-order kinetics, are evident in both the near- and far-ultraviolet circular dichroism of these enzymes. Gel filtration studies of inactivated subtilisin enzymes reveal the circular dichroism changes to be accompanied by the ap-pearance of aggregated protein material. The changes in circular dichroism and the production of associated subtilisin species are sensitive to protein concentration, denaturant concentrations, and pH. The circular dichroism of active subtilisins Novo and Carlsberg in guanidine hydrochloride exhibits irreversible changes similar to those observed for the inactivated subtilisins. Aggregated protein material is also formed initially in the presence of guanidine hydrochloride, but is rapidly autolyzed to low molecular weight fragments.