Andrew P Capaldi
- Associate Professor, Molecular and Cellular Biology
- Associate Professor, Genetics - GIDP
- Associate Professor, BIO5 Institute
- Distinguished Early Career Teaching Award
- College of Science, University of Arizona, Fall 2015
No activities entered.
DissertationMCB 920 (Spring 2018)
Internship in Applied BiosciABS 593A (Spring 2018)
Lab Presentations & DiscussionMCB 696A (Spring 2018)
DissertationMCB 920 (Fall 2017)
Introduction to ResearchMCB 795A (Fall 2017)
Lab Presentations & DiscussionMCB 696A (Fall 2017)
Master's ReportABS 909 (Summer I 2017)
DissertationMCB 920 (Spring 2017)
Introduction to ResearchMCB 795A (Spring 2017)
Lab Presentations & DiscussionMCB 696A (Spring 2017)
Master's ReportABS 909 (Spring 2017)
ResearchMCB 900 (Spring 2017)
DissertationMCB 920 (Fall 2016)
Integrative Approaches to BioMCB 585 (Fall 2016)
Internship in Applied BiosciABS 593A (Fall 2016)
Intro to Systems BiologyABE 580 (Fall 2016)
Intro to Systems BiologyMCB 480 (Fall 2016)
Intro to Systems BiologyMCB 580 (Fall 2016)
Introduction to ResearchMCB 795A (Fall 2016)
Lab Presentations & DiscussionMCB 696A (Fall 2016)
Lab Research RotationGENE 795A (Fall 2016)
ResearchMCB 900 (Fall 2016)
- Capaldi, A. P., Hughes Hallett, J., & Luo, X. (2015). Reversible Aggregation of the TOR Complex I protein Kog1 controls the threshold for growth initiation in budding yeast. eLife.
- Worley, J., Sullivan, A., Luo, X., Kaplan, M. E., & Capaldi, A. P. (2015). Genome-Wide Analysis of the TORC1 and Osmotic Stress Signaling Network in Saccharomyces cerevisiae. G3 (Bethesda, Md.).More infoThe Target of Rapamycin kinase Complex I (TORC1) is a master regulator of cell growth and metabolism in eukaryotes. Studies in yeast and human cells have shown that nitrogen/amino acid starvation signals act through Npr2/3 and the small GTPases Gtr1/2 (Rags in humans) to inhibit TORC1. However, it is unclear how other stress and starvation stimuli inhibit TORC1, and/or act in parallel with the TORC1 pathway, to control cell growth. To help answer these questions, we developed a novel automated pipeline and used it to measure the expression of a TORC1 dependent ribosome biogenesis gene (NSR1) during osmotic stress in 4700 Saccharomyces cerevisiae strains from the yeast knock-out collection. This led to the identification of 440 strains with significant and reproducible defects in NSR1 repression. The cell growth control and stress response proteins deleted in these strains form a highly connected network, including; 56 proteins involved in vesicle trafficking and vacuolar function; 53 proteins that act downstream of TORC1 according to a rapamycin assay--including components of the HDAC Rpd3L, Elongator, and the INO80, CAF-1 and SWI/SNF chromatin remodeling complexes; over 100 proteins involved in signaling and metabolism; and 17 proteins that directly interact with TORC1. These data provide an important resource for labs studying cell growth control and stress signaling, and demonstrate the utility of our new, and easily adaptable, method for mapping gene regulatory networks.
- Hughes Hallett, J. E., Luo, X., & Capaldi, A. P. (2014). State transitions in the TORC1 signaling pathway and information processing in Saccharomyces cerevisiae. Genetics, 198(2), 773-86.More infoTOR kinase complex I (TORC1) is a key regulator of cell growth and metabolism in all eukaryotes. Previous studies in yeast have shown that three GTPases-Gtr1, Gtr2, and Rho1-bind to TORC1 in nitrogen and amino acid starvation conditions to block phosphorylation of the S6 kinase Sch9 and activate protein phosphatase 2A (PP2A). This leads to downregulation of 450 Sch9-dependent protein and ribosome synthesis genes and upregulation of 100 PP2A-dependent nitrogen assimilation and amino acid synthesis genes. Here, using bandshift assays and microarray measurements, we show that the TORC1 pathway also populates three other stress/starvation states. First, in glucose starvation conditions, the AMP-activated protein kinase (AMPK/Snf1) and at least one other factor push the TORC1 pathway into an off state, in which Sch9-branch signaling and PP2A-branch signaling are both inhibited. Remarkably, the TORC1 pathway remains in the glucose starvation (PP2A inhibited) state even when cells are simultaneously starved for nitrogen and glucose. Second, in osmotic stress, the MAPK Hog1/p38 drives the TORC1 pathway into a different state, in which Sch9 signaling and PP2A-branch signaling are inhibited, but PP2A-branch signaling can still be activated by nitrogen starvation. Third, in oxidative stress and heat stress, TORC1-Sch9 signaling is blocked while weak PP2A-branch signaling occurs. Together, our data show that the TORC1 pathway acts as an information-processing hub, activating different genes in different conditions to ensure that available energy is allocated to drive growth, amino acid synthesis, or a stress response, depending on the needs of the cell.
- Worley, J., Luo, X., & Capaldi, A. P. (2013). Inositol Pyrophosphates Regulate Cell Growth and the Environmental Stress Response by Activating the HDAC Rpd3L. Cell Reports, 3(5), 1476-1482.More infoPMID: 23643537;PMCID: PMC3672359;Abstract: Cells respond to stress and starvation by adjusting their growth rate and enacting stress defense programs. In eukaryotes this involves inactivation of TORC1, which in turn triggers downregulation of ribosome and protein synthesis genes and upregulation of stress response genes. Here we report that the highly conserved inositol pyrophosphate (PP-IP) second messengers (including 1-PP-IP5, 5-PP-IP4, and 5-PP-IP5) are also critical regulators of cell growth and the general stress response, acting in parallel with the TORC1 pathway to control the activity of the class I histone deacetylase Rpd3L. In fact, yeast cells that cannot synthesize any of the PP-IPs mount little to no transcriptional response to osmotic, heat, or oxidative stress. Furthermore, PP-IP-dependent regulation of Rpd3L occurs independently of the role individual PP-IPs (such as 5-PP-IP5) play in activating specialized stress/starvation response pathways. Thus, the PP-IP second messengers simultaneously activate and tune the global response to stress and starvation signals. © 2013 The Authors.
- Buchan, J. R., Capaldi, A. P., & Parker, R. (2012). TOR-tured Yeast Find a New Way to Stand the Heat. Molecular Cell, 47(2), 155-157.More infoPMID: 22841000;Abstract: In this issue, Takahara and Maeda (2012) discover that together, Pbp1 and sequestration of the TORC1 complex in cytoplasmic mRNP stress granules provides a negative regulatory mechanism for TORC1 signaling during stress. © 2012 Elsevier Inc.
- Capaldi, A. P. (2010). Analysis of gene function using DNA microarrays. Methods in Enzymology, 470(C), 3-17.More infoPMID: 20946804;Abstract: This chapter provides a guide to analyzing gene function using DNA microarrays. First, I discuss the design and interpretation of experiments where gene expression levels in mutant and wild-type strains are compared. I then provide a detailed description of the protocols for isolating mRNA from yeast cells, converting the RNA into dye-labeled cDNA, and hybridizing these samples to a microarray. Finally, I discuss methods for washing, scanning, and analyzing the arrays. Emphasis is placed on describing approaches and techniques that help to minimize the artifacts and noise that so often plague microarray data. © 2010 Elsevier Inc. All rights reserved.
- Capaldi, A., & Capaldi, A. P. (2010). Analysis of gene function using DNA microarrays. Methods in enzymology, 470.More infoThis chapter provides a guide to analyzing gene function using DNA microarrays. First, I discuss the design and interpretation of experiments where gene expression levels in mutant and wild-type strains are compared. I then provide a detailed description of the protocols for isolating mRNA from yeast cells, converting the RNA into dye-labeled cDNA, and hybridizing these samples to a microarray. Finally, I discuss methods for washing, scanning, and analyzing the arrays. Emphasis is placed on describing approaches and techniques that help to minimize the artifacts and noise that so often plague microarray data.
- Capaldi, A. P., Kaplan, T., Liu, Y., Habib, N., Regev, A., Friedman, N., & O'Shea, E. K. (2008). Structure and function of a transcriptional network activated by the MAPK Hog1. Nature Genetics, 40(11), 1300-1306.More infoPMID: 18931682;PMCID: PMC2825711;Abstract: Cells regulate gene expression using a complex network of signaling pathways, transcription factors and promoters. To gain insight into the structure and function of these networks, we analyzed gene expression in single- and multiple-mutant strains to build a quantitative model of the Hog1 MAPK-dependent osmotic stress response in budding yeast. Our model reveals that the Hog1 and general stress (Msn2/4) pathways interact, at both the signaling and promoter level, to integrate information and create a context-dependent response. This study lays out a path to identifying and characterizing the role of signal integration and processing in other gene regulatory networks. © 2008 Nature Publishing Group.
- Capaldi, A. P., Kaplan, T., Liu, Y., Habib, N., Regev, A., Friedman, N., & O'Shea, E. K. (2008). Structure and function of a transcriptional network activated by the MAPK Hog1. Nature Genetics.More infoAbstract: Cells regulate gene expression using a complex network of signaling pathways, transcription factors and promoters. To gain insight into the structure and function of these networks, we analyzed gene expression in single- and multiple-mutant strains to build a quantitative model of the Hog1 MAPK-dependent osmotic stress response in budding yeast. Our model reveals that the Hog1 and general stress (Msn2/4) pathways interact, at both the signaling and promoter level, to integrate information and create a context-dependent response. This study lays out a path to identifying and characterizing the role of signal integration and processing in other gene regulatory networks.
- Gorski, S. A., S., C., Capaldi, A. P., Kalverda, A. P., Beddard, G. S., Moore, G. R., & Radford, S. E. (2004). Equilibrium Hydrogen Exchange Reveals Extensive Hydrogen Bonded Secondary Structure in the On-pathway Intermediate of Im7. Journal of Molecular Biology, 337(1), 183-193.More infoPMID: 15001361;Abstract: The four-helical immunity protein Im7 folds through an on-pathway intermediate that has a specific, but partially misfolded, hydrophobic core. In order to gain further insight into the structure of this species, we have identified the backbone hydrogen bonds formed in the ensemble by measuring the amide exchange rates (under EX2 conditions) of the wild-type protein and a variant, I72V. In this mutant the intermediate is significantly destabilised relative to the unfolded state (ΔΔGui=4.4kJ/mol), but the native state is only slightly destabilised (ΔΔG nu=1.8kJ/mol) at 10°C in 2H2O, pH* 7.0 containing 0.4 M Na2SO4, consistent with the view that this residue forms significant non-native stabilising interactions in the intermediate state. Comparison of the hydrogen exchange rates of the two proteins, therefore, enables the state from which hydrogen exchange occurs to be identified. The data show that amides in helices I, II and IV in both proteins exchange slowly with a free energy similar to that associated with global unfolding, suggesting that these helices form highly protected hydrogen-bonded helical structure in the intermediate. By contrast, amides in helix III exchange rapidly in both proteins. Importantly, the rate of exchange of amides in helix III are slowed substantially in the Im7* variant, I72V, compared with the wild-type protein, whilst other amides exchange more rapidly in the mutant protein, in accord with the kinetics of folding/unfolding measured using chevron analysis. These data demonstrate, therefore, that local fluctuations do not dominate the exchange mechanism and confirm that helix III does not form stable secondary structure in the intermediate. By combining these results with previously obtained Φ-values, we show that the on-pathway folding intermediate of Im7 contains extensive, stable hydrogen-bonded structure in helices I, II and IV, and that this structure is stabilised by both native and non-native interactions involving amino acid side-chains in these helices. © 2004 Elsevier Ltd. All rights reserved.
- Spence, G. R., Capaldi, A. P., & Radford, S. E. (2004). Trapping the on-pathway folding intermediate of Im7 at equilibrium. Journal of Molecular Biology, 341(1), 215-226.More infoPMID: 15312774;Abstract: The four-helical protein Im7 folds via a rapidly formed on-pathway intermediate (kUI=3000 s-1 at pH 7.0, 10 °C) that contains three (helices I, II and IV) of the four native α-helices. The relatively slow (kIN=300 s-1) conversion of this intermediate into the native structure is driven by the folding and docking of the six residue helix III onto the developing hydrophobic core. Here, we describe the structural properties of four Im7* variants designed to trap the protein in the intermediate state by disrupting the stabilising interactions formed between helix III and the rest of the protein structure. In two of these variants (I54A and L53AI54A), hydrophobic residues within helix III have been mutated to alanine, whilst in the other two mutants the sequence encompassing the native helix III was replaced by a glycine linker, three (H3G3) or six (H3G6) residues in length. All four variants were shown to be monomeric, as judged by analytical ultracentrifugation, and highly helical as measured by far-UV CD. In addition, all the variants denature co-operatively and have a stability (ΔGUF) and buried hydrophobic surface area (M UF) similar to those of the on-pathway kinetic intermediate. Structural characterisation of these variants using 1-anilino-8-napthalene sulphonic acid (ANS) binding, near-UV CD and 1D 1H NMR demonstrate further that the trapped intermediate ensemble is highly structured with little exposed hydrophobic surface area. Interestingly, however, the structural properties of the variants I54A and L53AI54A differ in detail from those of H3G3 and H3G6. In particular, the single tryptophan residue, located near the end of helix IV, and distant from helix III, is in a distinct environment in the two sets of mutants as judged by fluorescence, near-UV CD and the sensitivity of tryptophan fluorescence to iodide quenching. Overall, the results confirm previous kinetic analysis that demonstrated the hierarchical folding of Im7 via an on-pathway intermediate, and show that this species is a highly helical ensemble with a well-formed hydrophobic core. By contrast with the native state, however, the intermediate ensemble is flexible enough to change in response to mutation, its structural properties being tailored by residues in the sequence encompassing the native helix III. © 2004 Elsevier Ltd. All rights reserved.
- Friel, C. T., Capaldi, A. P., & Radford, S. E. (2003). Structural analysis of the rate-limiting transition states in the folding of Im7 and Im9: Similarities and differences in the folding of homologous proteins. Journal of Molecular Biology, 326(1), 293-305.More infoPMID: 12547210;Abstract: The bacterial immunity proteins Im7 and Im9 fold with mechanisms of different kinetic complexity. Whilst Im9 folds in a two-state transition at pH 7.0 and 10°C, Im7 populates an on-pathway intermediate under these conditions. In order to assess the role of sequence versus topology in the folding of these proteins, and to analyse the effect of populating an intermediate on the landscape for folding, we have determined the conformational properties of the rate-limiting transition state for Im9 folding/unfolding using ΦF-value analysis and have compared the results with similar data obtained previously for Im7. The data show that the rate-limiting transition states for Im9 and Im7 folding/unfolding are similar: both are compact (βT=0.94 and 0.89, respectively) and contain three of the four native helices docked around a specific hydrophobic core. Significant differences are observed, however, in the magnitude of the ΦF-values obtained for the two proteins. Of the 20 residues studied in both proteins, ten have ΦF-values in Im7 that exceed those in Im9 by more than 0.2, and of these five differ by more than 0.4. The data suggest that the population of an intermediate in Im7 results in folding via a transition state ensemble that is conformationally restricted relative to that of Im9. The data are consistent with the view that topology is an important determinant of folding. Importantly, however, they also demonstrate that while the folding transition state may be conserved in homologous proteins that fold with two and three-state kinetics, the population of an intermediate can have a significant effect on the breadth of the transition state ensemble. © 2003 Elsevier Science Ltd. All rights reserved.
- Capaldi, A. P., Kleanthous, C., & Radford, S. E. (2002). Im7 folding mechanism: Misfolding on a path to the native state. Nature Structural Biology, 9(3), 209-216.More infoPMID: 11875516;Abstract: Many proteins populate collapsed intermediate states during folding. In order to elucidate the nature and importance of these species, we have mapped the structure of the on-pathway intermediate of the four-helix protein, Im7, together with the conformational changes it undergoes as it folds to the native state. Kinetic data for 29 Im7 point mutants show that the intermediate contains three of the four helices found in the native structure, packed around a specific hydrophobic core. However, the intermediate contains many non-native interactions; as a result, hydrophobic interactions become disrupted in the rate-limiting transition state before the final helix docks onto the developing structure. The results of this study support a hierarchical mechanism of protein folding and explain why the misfolding of Im7 occurs. The data also demonstrate that non-native interactions can play a significant role in folding, even for small proteins with simple topologies.
- Capaldi, A. P., & Radford, S. E. (2001). An unfolding story. Trends in Biochemical Sciences, 26(12), 753-.
- Capaldi, A. P., C., M., Kleanthous, C., Roder, H., & Radford, S. E. (2001). Ultrarapid mixing experiments reveal that Im7 folds via an on-pathway intermediate. Nature Structural Biology, 8(1), 68-72.More infoPMID: 11135674;Abstract: Many proteins populate partially organized structures during folding. Since these intermediates often accumulate within the dead time (2-5 ms) of conventional stopped-flow and quench-flow devices, it has been difficult to determine their role in the formation of the native state. Here we use a microcapillary mixing apparatus, with a time resolution of ∼150 μs, to directly follow the formation of an intermediate in the folding of a four-helix protein, Im7. Quantitative kinetic modeling of folding and unfolding data acquired over a wide range of urea concentrations demonstrate that this intermediate ensemble lies on a direct path from the unfolded to the native state.
- Ferguson, N., Wei, L. i., Capaldi, A. P., Kleanthous, C., & Radford, S. E. (2001). Using chimeric immunity proteins to explore the energy landscape for α-helical protein folding. Journal of Molecular Biology, 307(1), 393-405.More infoPMID: 11243827;Abstract: To address the role of sequence in the folding of homologous proteins, the folding and unfolding kinetics of the all-helical bacterial immunity proteins Im2 and Im9 were characterised, together with six chimeric derivatives of these proteins. We show that both Im2 and Im9 fold rapidly (kUNH2O ≈ 2000 s-1 at pH 7.0, 25°C) in apparent two-state transitions, through rate-limiting transition states that are highly compact (βTS 0.93 and 0.96, respectively). Whilst the folding and unfolding properties of three of the chimeras (Im2 (1-44)Im9, Im2 (1-64)Im9 and Im2 (25-44)Im9) are similar to their parental counterparts, in other chimeric proteins the introduced sequence variation results in altered kinetic behaviour. At low urea concentrations, Im2 (1-29)Im9 and Im2 (56-64)Im9 fold in two-state transitions via transition states that are significantly less compact (βTS ≈ 0.7) than those characterised for the other immunity proteins presented here. At higher urea concentrations, however, the rate-limiting transition state for these two chimeras switches or moves to a more compact species (βTS ≈ 0.9). Surprisingly, Im2 (30-64)Im9 populates a highly collapsed species (βI = 0.87) in the dead-time (2.5 ms) of stopped flow measurements. These data indicate that whilst topology may place significant constraints on the folding process, specific inter-residue interactions, revealed here through multiple sequence changes, can modulate the ruggedness of the folding energy landscape. © 2001 Academic Press.
- Gorski, S. A., Capaldi, A. P., Kleanthous, C., & Radford, S. E. (2001). Acidic conditions stabilise intermediates populated during the folding of Im7 and Im9. Journal of Molecular Biology, 312(4), 849-863.More infoPMID: 11575937;Abstract: The helical bacterial immunity proteins Im7 and Im9 have been shown to fold via kinetic mechanisms of differing complexity, despite having 60% sequence identity. At pH 7.0 and 10°C, Im7 folds in a three-state mechanism involving an on-pathway intermediate, while Im9 folds in an apparent two-state transition. In order to examine the folding mechanisms of these proteins in more detail, the folding kinetics of both Im7 and Im9 (at 10°C in 0.4 M sodium sulphate) have been examined as a function of pH. Kinetic modelling of the folding and unfolding data for Im7 between pH 5.0 and 8.0 shows that the on-pathway intermediate is stabilised by more acidic conditions, whilst the native state is destabilised. The opposing effect of pH on the stability of these states results in a significant population of the intermediate at equilibrium at pH 6.0 and below. At pH 7.0, the folding and unfolding kinetics for Im9 can be fitted adequately by a two-state model, in accord with previous results. However, under acidic conditions there is a clear change of slope in the plot of the logarithm of the folding rate constant versus denaturant concentration, consistent with the population of one or more intermediate(s) early during folding. The kinetic data for Im9 at these pH values can be fitted to a three-state model, where the intermediate ensemble is stabilised and the native state destabilised as the pH is reduced, rationalising previous results that showed that an intermediate is not observed experimentally at pH 7.0. The data suggest that intermediate formation is a general step in immunity protein folding and demonstrate that it is necessary to explore a wide range of refolding conditions in order to show that intermediates do not form in the folding of other small, single-domain proteins. © 2001 Academic Press.
- Shah, A. M., Conn, D. A., Li, S. -., Capaldi, A., Jäger, J., & Sweasy, J. B. (2001). A DNA polymerase β mutator mutant with reduced nucleotide discrimination and increased protein stability. Biochemistry, 40(38), 11372-11381.More infoPMID: 11560485;Abstract: DNA polymerase β (pol β) offers a simple system to examine the role of polymerase structure in the fidelity of DNA synthesis. In this study, the M282L variant of pol β (M282Lβ) was identified using an in vivo genetic screen. Met282, which does not contact the DNA template or the incoming deoxynucleoside triphosphate (dNTP) substrate, is located on α-helix N of pol β. This mutant enzyme demonstrates increased mutagenesis in both in vivo and in vitro assays. M282Lβ has a 7.5-fold higher mutation frequency than wild-type pol β; M282Lβ commits a variety of base substitution and frameshift errors. Transient-state kinetic methods were used to investigate the mechanism of intrinsic mutator activity of M282Lβ. Results show an 11-fold decrease in dNTP substrate discrimination at the level of ground-state binding. However, during the protein conformational change and/or phosphodiester bond formation, the nucleotide discrimination is improved. X-ray crystallography was utilized to gain insights into the structural basis of the decreased DNA synthesis fidelity. Most of the structural changes are localized to site 282 and the surrounding region in the C-terminal part of the 31-kDa domain. Repositioning of mostly hydrophobic amino acid residues in the core of the C-terminal portion generates a protein with enhanced stability. The combination of structural and equilibrium unfolding data suggests that the mechanism of nucleotide discrimination is possibly affected by the compacting of the hydrophobic core around residue Leu282. Subsequent movement of an adjacent surface residue, Arg283, produces a slight increase in volume of the pocket that may accommodate the incoming correct base pair. The structural changes of M282Lβ ultimately lead to an overall reduction in polymerase fidelity.
- Jones, S., Reader, J. S., Healy, M., Capaldi, A. P., Ashcroft, A. E., Kalverda, A. P., Smith, D. A., & Radford, S. E. (2000). Partially unfolded species populated during equilibrium denaturation of the β-sheet protein Y74W apo-pseudoazurin. Biochemistry, 39(19), 5672-5682.More infoPMID: 10801317;Abstract: Apo-pseudoazurin is a single domain cupredoxin. We have engineered a mutant in which a unique tryptophan replaces the tyrosine residue found in the tyrosine comer of this Greek key protein, a region that has been proposed to have an important role in folding. Equilibrium denaturation of Y74W apo- pseudoazurin demonstrated multistate unfolding in urea (pH 7.0, 0.5 M Na2SO4 at 15 °C), in which one or more partially folded species are populated in 4.3 M urea. Using a variety of biophysical techniques, we show that these species, on average, have lost a substantial portion of the native secondary structure, lack fixed tertiary packing involving tryptophan and tyrosine residues, are less compact than the native state as determined by fluorescence lifetimes and time-resolved anisotropy, but retain significant residual structure involving the trytophan residue. Peptides ranging in length from 11 to 30 residues encompassing this region, however, did not contain detectable nonrandom structure, suggesting that long-range interactions are important for stabilizing the equilibrium partially unfolded species in the intact protein. On the basis of these results, we suggest that the equilibrium denaturation of Y74W apo-pseudoazurin generates one or more partially unfolded species that are globally collapsed and retain elements of the native structure involving the newly introduced tryptophan residue. We speculate on the role of such intermediates in the generation of the complex Greek key fold.
- Capaldi, A. P., Ferguson, S. J., & Radford, S. E. (1999). The Greek key protein apo-pseudoazurin folds through an obligate on-pathway intermediate. Journal of Molecular Biology, 286(5), 1621-1632.More infoPMID: 10064719;Abstract: Folding of the 123 amino acid residue Greek key protein apo-pseudoazurin from Thiosphaera pantotropha has been examined using stopped-flow circular dichroism in 0.5 M Na2SO4 at pH 7.0 and 15°C. The data show that the protein folds from the unfolded state with all eight proline residues in their native isomers (seven trans and one cis) to an intermediate within the dead-time of the stopped-flow mixing (50 ms). The urea dependence of the rates of folding and unfolding of the protein were also determined. The ratio of the folding rate to the unfolding rate (extrapolated into water) is several orders of magnitude too small to account for the equilibrium stability of the protein, consistent with the population of an intermediate. Despite this, the logarithm of the rate of folding versus denaturant concentration is linear. These data can be rationalised by the population of an intermediate under all refolding conditions. Accordingly, kinetic and equilibrium measurements were combined to fit the chevron plot to an on-pathway model (U⇆I⇆N). The fit shows that apo-pseudoazurin rapidly forms a compact species that is stabilised by 25 kJ/mol before folding to the native state at a rate of 2 s-1. Although the data can also be fitted to an off-pathway model (I⇆U⇆N), the resulting kinetic parameters indicate that the protein would have to fold to the native state at a rate of 86,000 s-1 (a time constant of only 12 μs). Similarly, models in which this intermediate is bypassed also lead to unreasonably fast refolding rates. Thus, the intermediate populated during the refolding of apo-pseudoazurin appears to be obligate and on the folding pathway. We suggest, based on this study and others, that some intermediates play a critical role in limiting the search to the native state.
- Ferguson, N., Capaldi, A. P., James, R., Kleanthous, C., & Radford, S. E. (1999). Rapid folding with and without populated intermediates in the homologous four-helix proteins Im7 and Im9. Journal of Molecular Biology, 286(5), 1597-1608.More infoPMID: 10064717;Abstract: The kinetics and thermodynamics of the folding of the homologous four-helix proteins Im7 and Im9 have been characterised at PH 7.0 and 10°C. These proteins are 60% identical in sequence and have the same three-dimensional structure, yet appear to fold by different kinetic mechanisms. The logarithm of the folding and unfolding rates of Im9 change linearly as a function of urea concentration and fit well to an equation describing a two-state mechanism (with a folding rate of 1500 s-1, an unfolding rate of 0.01 s-1, and a highly compact transition state that has ~95% of the native surface area buried). By contrast, there is clear evidence for the population of an intermediate during the refolding of Im7, as indicated by a change in the urea dependence of the folding rate and the presence of a significant burst phase amplitude in the refolding kinetics. Under stabilising conditions (0.25 M Na2SO4, pH 7.0 and 10°C) the folding of Im9 remains two-state, whilst under similar conditions (0.4 M Na2SO4, pH 7.0 and 10°C) the intermediate populated during Im7 refolding is significantly stabilised (K(UI) = 125). Equilibrium denaturation experiments, under the conditions used in the kinetic measurements, show that Im7 is significantly less stable than Im9 (ΔΔG 9.3 kJ/mol) and the ΔG and m values determined accord with those obtained from the fit to the kinetic data. The results show, therefore, that the population of an intermediate in the refolding of the immunity protein structure is defined by the precise amino acid sequence rather than the global stability of the protein. We discuss the possibility that the intermediate of Im7 is populated due to differences in helix propensity in Im7 and Im9 and the relevance of these data to the folding of helical proteins in general.
- Capaldi, A. P., & Radford, S. E. (1998). Kinetic studies of β-sheet protein folding. Current Opinion in Structural Biology, 8(1), 86-92.More infoPMID: 9519300;Abstract: New studies have shown that folding of β-sheet proteins can occur with and without intermediates, with fast to slow refolding rates and late to very late transition states. These experiments demonstrate that, despite early speculation to the contrary, β-sheet protein folding does not appear to be fundamentally different from that of helical and mixed α,β proteins.