Folding and oxidation of the antibody domain C(H)3

The non-covalent homodimer formed by the C-terminal domains of the IgG1 heavy chains (C(H)3) is the simplest naturally occurring model system for studying immunoglobulin folding and assembly. In the native state, the intrachain disulfide bridge, which connects a three-stranded and a four-stranded beta-sheet is buried in the hydrophobic core of the protein. Here, we show that the disulfide bridge is not required for folding and association, since the reduced C(H)3 domain folds to a dimer with defined secondary and tertiary structure. However, the thermodynamic stability of the reduced C(H)3 dimer is much lower than that of the oxidized state. This allows the formation of disulfide bonds either concomitant with folding (starting from the reduced, denatured state) or after folding (starting from the reduced dimer). The analysis of the two processes revealed that, under all conditions investigated, one of the cysteine residues, Cys 86, reacts preferentially with oxidized glutathione to a mixed disulfide that subsequently interacts with the less-reactive second thiol group of the intra-molecular disulfide bond. For folded C(H)3, the second step in the oxidation process is slow. In contrast, starting from the unfolded and reduced protein, the oxidation reaction is faster. However, the overall folding reaction of C(H)3 during oxidative folding is a slow process. Especially, dimerization is slow, compared to the association starting from the denatured oxidized state. This deceleration may be due to misfolded conformations trapped by the disulfide bridge.     

Low energy pathways and non-native interactions: the influence of artificial disulfide bridges on the mechanism of folding

Four versions of a beta-sheet protein (CD2.d1) have been made, each with a single artificial disulfide bond inserted into hairpin structures. Folding kinetics of reduced and oxidized forms shows bridge position strongly influences its effect on the folding reaction. Bridging residues 58 and 62 does not affect the rapidly formed intermediate (I) or rate-limiting transition (t) state, whereas bridging 33 and 38, or 31 and 41, lowers the t-state energy, with the latter having the stronger influence. Bridging residues 79 and 90 stabilizes both I- and t-states. To assess additivity in the energetic effects of these bridges, four double-bridge variants have also been made. All show precise additivity of overall stability, with two showing additivity when ground states and the rate-limiting t-state are assessed, i.e. no measurable change in the folding mechanism occurs. However, combining 31-41 and 79-90 bridges produces a molecule that folds through a different pathway, with a much more stable intermediate than expected and a much higher t-state barrier. This is explained by the artificial introduction of stabilizing, non-native contacts in the I-state. More surprisingly, for another double-bridge version (58-62 and 79-90) both I- and t-states are less stable than expected, showing that conformational constraints introduced by the two bridges prevent formation of non-native contacts that would otherwise stabilize the I- and t-states, thereby lowering the energy of the folding landscape in the wild-type (unbridged) molecule. We conclude that the lowest energy path for folding has I- and t-state structures that are stabilized by non-native interactions.     

Hydrogen-deuterium exchange in free and prodomain-complexed subtilisin

Residue-specific exchange rates of 223 amide protons in free and prodomain-complexed subtilisin were determined in order to understand how the prodomain binding affects the energetics of subtilisin folding. In free subtilisin, amide protons can be categorized according to exchange rate: 74 fast exchangers (rates > or = 1 h(-1)); 52 medium exchangers (rates between 1 h(-1) and 1 day(-1)); 31 slow exchangers (rates between 1 day(-1) and 0.001 day(-1)). The remaining 66 amide proteins did not exchange detectibly over 9 months (k(obs) < year(-1)) and were denoted as core protons. Core residues occur throughout the main structural elements of subtilisin. Prodomain binding results in high protection factors (100-1000) in the central beta-sheet, particularly in the vicinity of beta-strands S5, S6, and S7 and the connecting loops between them. These connecting loops provide the ligands to the cation at metal site B. Overall, prodomain binding seems to facilitate the organization of the entire central beta-sheet and alpha-helix C in the left-handed crossover connection between beta-strands two and three. It also appears to facilitate the isomerization of multiple prolines late in folding, allowing the formation of metal site B. The gain of stability region around site B comes at the cost of stability in regions more distal to prodomain binding: the C-terminal alpha-helix H and the N-terminal alpha-helices A and B. The acceleration of exchange in these regions by prodomain binding reveals an antagonism between the folding intermediate and the full native structure. This antagonism helps to explain why the prodomain is needed to stabilize the folding intermediate as well as why the unfolding of free subtilisin seldom occurs via this intermediate.     

Contribution of disulfide bonds to stability of the folding intermediate of alpha-lactalbumin

The secondary structure formed in disulfide reduced alpha-lactalbumin is investigated by CD spectrum and is compared with that of the folding intermediate of the disulfide intact protein. The peptide backbone structure of the reduced protein depends strongly on salt concentration in contrast to that of the intermediate. It is close to a random coil in the absence of salt, but it is almost the same as that of the intermediate at a high concentration of salt. The secondary structures of both the proteins undergo broad unfolding transitions when temperature is raised or when urea is added. The secondary structure of the reduced protein is less stable against both heat and urea. These results show that the disulfide bonds are not a determinant of the secondary structure formed at an early stage of folding, and they stabilize the secondary structure of the folding intermediate.     

Evidence for the underlying cause of diversity of the disulfide folding pathway

The pathways of oxidative folding of disulfide proteins exhibit a high degree of diversity, which is illustrated by the varied extent of (a) the heterogeneity of folding intermediates, (b) the predominance of intermediates containing native disulfide bonds, and (c) the level of accumulation of fully oxidized scrambled isomers as intermediates. BPTI and hirudin exemplify two extreme cases of such divergent folding pathways. We previously proposed that the underlying cause of this diversity is associated with the degree of stability of protein subdomains. Here we present compelling evidence that substantiates this hypothesis by studying the folding pathway of alphaLA-IIA. alphaLA-IIA is a partially folded intermediate of alpha-lactalbumin (alphaLA). It comprises a structured beta-sheet (calcium-binding) domain linked by two native disulfide bonds (Cys(61)-Cys(77) and Cys(73)-Cys(91)) and a disordered alpha-helical domain with four free cysteines (Cys(6), Cys(28), Cys(111), and Cys(120)). Purified alphaLA-IIA was allowed to refold without and with stabilization of its structured beta-sheet domain by calcium. In the absence of calcium, the folding pathway of alphaLA-IIA resembles that of hirudin, displaying a highly heterogeneous population of folding intermediates, including fully oxidized scrambled species. Upon stabilization of its beta-sheet domain by bound calcium, oxidative folding of alphaLA-IIA undergoes a pathway conspicuously similar to that of BPTI, exhibiting limited species of folding intermediates containing mostly native disulfide bonds.     

Understanding the contribution of disulfide bridges to the folding and misfolding of an anti‐Aβ scFv

ScFv‐h3D6 is a single chain variable fragment that precludes Aβ peptide‐induced cytotoxicity by withdrawing Aβ oligomers from the amyloid pathway to the worm‐like pathway. Production of scFv molecules is not a straightforward procedure because of the occurrence of disulfide scrambled conformations generated in the refolding process. Here, we separately removed the disulfide bond of each domain and solved the scrambling problem; and then, we intended to compensate the loss of thermodynamic stability by adding three C‐terminal elongation mutations, previously described to stabilize the native fold of scFv‐h3D6. Such stabilization occurred through stabilization of the intermediate state in the folding pathway and destabilization of a different, β‐rich, intermediate state driving to worm‐like fibrils. Elimination of the disulfide bridge of the less stable domain, V_L, deeply compromised the yield and increased the aggregation tendency, but elimination of the disulfide bridge of the more stable domain, V_H, solved the scrambling problem and doubled the production yield. Notably, it also changed the aggregation pathway from the protective worm‐like morphology to an amyloid one. This was so because a partially unfolded intermediate driving to amyloid aggregation was present, instead of the β‐rich intermediate driving to worm‐like fibrils. When combining with the elongation mutants, stabilization of the partially unfolded intermediate driving to amyloid fibrils was the only effect observed. Therefore, the same mutations drove to completely different scenarios depending on the presence of disulfide bridges and this illustrates the relevance of such linkages in the stability of different intermediate states for folding and misfolding.     

Mapping the interactions present in the transition state for unfolding/folding of FKBP12.

The structure of the transition state for folding/unfolding of the immunophilin FKBP12 has been characterised using a combination of protein engineering techniques, unfolding kinetics, and molecular dynamics simulations. A total of 34 mutations were made at sites throughout the protein to probe the extent of secondary and tertiary structure in the transition state. The transition state for folding is compact compared with the unfolded state, with an approximately 30 % increase in the native solvent-accessible surface area. All of the interactions are substantially weaker in the transition state, as probed by both experiment and molecular dynamics simulations. In contrast to some other proteins of this size, no element of structure is fully formed in the transition state; instead, the transition state is similar to that found for smaller, single-domain proteins, such as chymotrypsin inhibitor 2 and the SH3 domain from alpha-spectrin. For FKBP12, the central three strands of the beta-sheet, beta-strand 2, beta-strand 4 and beta-strand 5, comprise the most structured region of the transition state. In particular Val101, which is one of the most highly buried residues and located in the middle of the central beta-strand, makes approximately 60 % of its native interactions. The outer beta-strands and the ends of the central beta-strands are formed to a lesser degree. The short alpha-helix is largely unstructured in the transition state, as are the loops. The data are consistent with a nucleation-condensation model of folding, the nucleus of which is formed by side-chains within beta-strands 2, 4 and 5, and the C terminus of the alpha-helix. The precise residues involved in the nucleus differ in the two simulated transition state ensembles, but the interacting regions of the protein are conserved. These residues are distant in the primary sequence, demonstrating the importance of tertiary interactions in the transition state. The two independently derived transition state ensembles are structurally similar, which is consistent with a Bronsted analysis confirming that the transition state is an ensemble of states close in structure.     

The role of a disulfide bridge in the stability and folding kinetics of Arabidopsis thaliana cytochrome c(6A)

Cytochrome c(6A) is a eukaryotic member of the Class I cytochrome c family possessing a high structural homology with photosynthetic cytochrome c(6) from cyanobacteria, but structurally and functionally distinct through the presence of a disulfide bond and a heme mid-point redox potential of +71mV (vs normal hydrogen electrode). The disulfide bond is part of a loop insertion peptide that forms a cap-like structure on top of the core α-helical fold. We have investigated the contribution of the disulfide bond to thermodynamic stability and (un)folding kinetics in cytochrome c(6A) from Arabidopsis thaliana by making comparison with a photosynthetic cytochrome c(6) from Phormidium laminosum and through a mutant in which the Cys residues have been replaced with Ser residues (C67/73S). We find that the disulfide bond makes a significant contribution to overall stability in both the ferric and ferrous heme states. Both cytochromes c(6A) and c(6) fold rapidly at neutral pH through an on-pathway intermediate. The unfolding rate for the C67/73S variant is significantly increased indicating that the formation of this region occurs late in the folding pathway. We conclude that the disulfide bridge in cytochrome c(6A) acts as a conformational restraint in both the folding intermediate and native state of the protein and that it likely serves a structural rather than a previously proposed catalytic role.     

Structural trees for proteins containing φ-motifs

In the present study, a novel structural motif of proteins referred to as the phi-motif is considered, and two novel structural trees in which the phi-motif is taken as the root structure have been constructed. The simplest phi-motif is formed by three adjacent beta-strands connected by loops and packed in one beta-sheet so that its overall fold resembles the Greek letter phi. Construction of the structural trees and modeling of folding pathways have shown that all structures of the protein superfamilies can be obtained by stepwise addition of alpha-helices and/or beta-strands to the root phi-motif taking into account a restricted set of rules inferred from known principles of protein structure. The structural trees are a good tool for structure comparison, structural classification of proteins, as well as for searching for all possible protein folds and folding pathways.     

Preferential binding of an unfolded protein to DsbA.

The oxidoreductase DsbA from the periplasm of escherichia coli introduces disulfide bonds into proteins at an extremely high rate. During oxidation, a mixed disulfide is formed between DsbA and the folding protein chain, and this covalent intermediate reacts very rapidly either to form the oxidized protein or to revert back to oxidized DsbA. To investigate its properties, a stable form of the intermediate was produced by reacting the C33A variant of DsbA with a variant of RNase T1. We find that in this stable mixed disulfide the conformational stability of the substrate protein is decreased by 5 kJ/mol, whereas the conformational stability of DsbA is increased by 5 kJ/mol. This reciprocal effect suggests strongly that DsbA interacts with the unfolded substrate protein not only by the covalent disulfide bond, but also by preferential non-covalent interactions. The existence of a polypeptide binding site explains why DsbA oxidizes protein substrates much more rapidly than small thiol compounds. Such a very fast reaction is probably important for protein folding in the periplasm, because the accessibility of the thiol groups for DsbA can decrease rapidly when newly exported polypeptide chains begin to fold.     

Comparison of local and global stability of an analogue of a disulfide-folding intermediate with those of the wild-type protein in bovine pancreatic ribonuclease A: identification of specific regions of stable structure along the oxidative folding pathway

We have identified specific regions of the polypeptide chain of bovine pancreatic ribonuclease A (RNase A) that are critical for stabilizing the oxidative folding intermediate des-[40-95] (with three native disulfide bonds but lacking the fourth native Cys40-Cys95 disulfide bond) in an ensemble of largely disordered three-disulfide precursors (3S if des-[40-95]). A stable analogue of des-[40-95], viz., [C40A, C95A] RNase A, which contains three out of four native disulfide pairings, was previously found to have a three-dimensional structure very similar to that of the wild-type protein. However, it is determined here from GdnHCl denaturation experiments to have significantly reduced global stability, i.e., = 4.5 kcal /mol at 20 degrees C and pH 4.6. The local stability of [C40A, C95A] RNase A was also examined using site-specific amide (2)H/(1)H exchange measurements at pD 5.0 to determine the individual unfolding free energy of specific residues under both strongly native (12 degrees C) and more destabilizing (20 degrees C) conditions. Comparison of the relative stabilities at specific amide sites of [C40A, C95A] RNase A at both temperatures with the corresponding values for the wild-type protein at 35 degrees C corroborates previous experimental evidence that unidentified intramolecular contacts in the vicinity of the preferentially formed native one-disulfide (C65-C72) loop are crucial for stabilizing early folding intermediates, leading to des-[40-95]. Moreover, values of for residues at or near the third alpha-helix, and in part of the second beta-sheet of [C40A, C95A] RNase A, indicate that these two regions of regular backbone structure contribute to stabilizing the global chain fold of the des-[40-95] disulfide-folding intermediate in the wild-type protein. More significantly, we have identified numerous specific residues in the first alpha-helix and the first beta-sheet of the protein that are stabilized in the final step of the major oxidative regeneration pathway of RNase A (des-[40-95] --> N).     

Mechanism of the kinetically-controlled folding reaction of subtilisin

Like many secreted proteases, subtilisin is kinetically stable in the mature form but unable to fold without assistance from its prodomain. The existence of high kinetic barriers to folding challenges many widely accepted ideas, namely, the thermodynamic determination of native structure and the sufficiency of thermodynamic stability to determine a pathway. The purpose of this article is to elucidate the physical nature of the kinetic barriers to subtilisin folding and to show how the prodomain overcomes these barriers. To address these questions, we have studied the bimolecular folding reaction of the subtilisin prodomain and a series of subtilisin mutants, which were designed to explore the steps in the folding reaction. Our analysis shows that inordinately slow folding of the mature form of subtilisin results from the accrued effects of two slow and sequential processes: (1) the formation of an unstable and topologically challenged intermediate and (2) the proline-limited isomerization of the intermediate to the native state. The low stability of nascent folding intermediates results in part from subtilisin's high dependence on metal binding for stability. Native subtilisin is thermodynamically unstable in the absence of bound metals. Because the two metal binding sites are formed late in folding, however, they contribute little to the stability of folding intermediates. The formation of productive folding intermediates is further hindered by the topological challenge of forming a left-handed crossover connection between beta-strands S2 and S3. This connection is critical to propagate the folding reaction. In the presence of the prodomain, folding proceeds through one major intermediate, which is stabilized by prodomain binding, independent of metal concentration and proline isomerization state. The prodomain also catalyzes the late proline isomerizations needed to form metal site B. Rate-limiting proline isomerization is common in protein folding, but its effect in slowing subtilisin folding is amplified because of the instability of the intermediate and an apparent need for simultaneous isomerization of multiple prolines in order to create metal site B. Thus, the kinetically controlled folding reaction of subtilisin, although unusual, is explained by the accrued effects of events found in other proteins.     

Heterodimer formation between thioredoxin f and fructose 1,6-bisphosphatase from spinach chloroplasts

Chloroplast fructose 1,6-bisphosphatase (FBPase) is activated by reduction of a regulatory disulfide through thioredoxin f (Trx f). In the course of this reduction a transient mixed disulfide is formed linking covalently Trx f with FBPase, which possesses three Cys on a loop structure, two of them forming the redox-active disulfide bridge. The goal of this study was to identify the Cys involved in the transient mixed disulfide. To stabilize this reaction intermediate, mutant proteins with modified active sites were used. We identified Cys-155 of the FBPase as the one engaged in the formation of the mixed disulfide intermediate with Cys-46 of Trx f.     

A comparison of folding techniques in the chemical synthesis of the epidermal growth factor-like domain in neu differentiation factor alpha/beta.

The 52-residue alpha/beta chimera of the epidermal growth factor-like domain in neu differentiation factor (NDFealpha/beta) has been synthesized and folded to form a three disulfide bridge (Cys182-Cys196, Cys190-Cys210, Cys212-Cys221) containing peptide. We investigated two general strategies for the formation of the intramolecular disulfide bridges including, the single-step approach, which used fully deprotected and reduced peptide, and a sequential approach that relied on orthogonal cysteine protection in which specific pairs are excluded from the first oxidation step. Because there are 15 possible disulfide bridge arrangements in a peptide with six cysteines, the one-step approach may not always provide the desired disulfide pairing. Here, we compare the single-step approach with a systematic evaluation of the sequential approach. We employed the acetamidomethyl group to protect each pair of cysteines involved in disulfide bridges, i.e. Cys182 to Cys196, Cys190 to Cys210 and Cys212 to Cys221. This reduced the number of possible disulfide patterns from 15 to three in the first folding step. We compared the efficiencies of folding for each protected pair using RP-HPLC, mapped the disulfide connectivity of the predominant product and then formed the final disulfide from the partially folded intermediate via 12 oxidation. Only the peptide having the Cys182-Cys196 pair blocked with acetamidomethyl forms the desired disulfide isomer (Cys190-Cys210/Cys212-Cys221) as a single homogeneous product. By optimizing both approaches, as well as other steps in the synthesis, we can now rapidly provide large-scale syntheses of NDFealpha/beta and other novel EGF-like peptides.     

Calorimetric dissection of thermal unfolding of OspA,a predominantly beta-sheet protein containing a single-layer beta-sheet

Outer surface protein A (OspA) from Borrelia burgdorferi is a predominantly beta-sheet protein comprised of beta-strands beta1-beta21 and a short C-terminal alpha-helix. It contains two globular domains (N and C-terminal domains) and a unique single-layer beta-sheet (central beta-sheet) that connects the two domains. OspA contains an unusually large number of charged amino acid residues. To understand the mechanism of stabilization of this unique beta-sheet protein, thorough thermodynamic investigations of OspA and its truncated mutant lacking a part of the C-terminal domain were conducted using calorimetry and circular dichroism. The stability of OspA was found to be sensitive to pH and salt concentration. The heat capacity curve clearly consisted of two components, and all the thermodynamic parameters were obtained for each step. The thermodynamic parameters associated with the two transitions are consistent with a previously proposed model, in which the first transition corresponds to the unfolding of the C-terminal domain and the last two beta-strands of the central beta-sheet, and the second transition corresponds to that of the N-terminal domain and the first beta-strand of the central beta-sheet in the second peak. The ratio of calorimetric and van't Hoff enthalpies indicates that the first peak includes another thermodynamic intermediate state. Large heat capacity changes were observed for both transitions, indicative of large changes in the exposure of hydrophobic surfaces associated with the transitions. This observation demonstrates that hydrophobic parts are buried efficiently in the native structure in spite of the low content of hydrophobic residues in OspA. By decomposing the enthalpy, entropy, and Gibbs free energy into contributions from different interactions, we found that the enthalpy changes for hydrogen bonding and polar interactions are exceptionally large, indicating that OspA maintains its stability by making full use of its unique beta-sheet and high content of polar residues. These thermodynamic analyses demonstrated that it is possible to maintain protein tertiary structure by making effective use of an unusual amino acid composition.     

Effect of an alternative disulfide bond on the structure, stability, and folding of human lysozyme

Human lysozyme has four disulfide bonds, one of which, Cys65-Cys81, is included in a long loop of the beta-domain. A cysteine-scanning mutagenesis in which the position of Cys65 was shifted within a continuous segment from positions 61 to 67, with fixed Cys81, has previously shown that only the mutant W64CC65A, which has a nonnative Cys64-Cys81 disulfide, can be correctly folded and secreted by yeast. Here, using the W64CC65A mutant, we investigated the effects of an alternative disulfide bond on the structure, stability, and folding of human lysozyme using circular dichroism (CD) and fluorescence spectroscopy combined with a stopped-flow technique. Although the mutant is expected to have a different main-chain structure from that of the wild-type protein around the loop region, far- and near-UV CD spectra show that the native state of the mutant has tightly packed side chains and secondary structure similar to that of the wild-type. Guanidine hydrochloride-induced equilibrium unfolding transition of the mutant is reversible, showing high stability and cooperativity of folding. In the kinetic folding reaction, both proteins accumulate a similar burst-phase intermediate having pronounced secondary structure within the dead time of the measurement and fold into the native structure by means of a similar folding mechanism. Both the kinetic refolding and unfolding reactions of the mutant protein are faster than those of the wild-type, but the increase in the unfolding rate is larger than that of the refolding rate. The Gibbs' free-energy diagrams obtained from the kinetic analysis suggest that the structure around the loop region in the beta-domain of human lysozyme is formed after the transition state of folding, and thus, the effect of the alternative disulfide bond on the structure, stability, and folding of human lysozyme appears mainly in the native state.     

Anabaena apoflavodoxin hydrogen exchange: on the stable exchange core of the alpha/beta(21345) flavodoxin-like family

An important issue in modern protein biophysics is whether structurally homologous proteins share common stability and/or folding features. Flavodoxin is an archetypal alpha/beta protein organized in three layers: a central beta-sheet (strand order 21345) flanked by helices 1 and 5 on one side and helices 2, 3, and 4 on the opposite side. The backbone internal dynamics of the apoflavodoxin from Anabaena is analyzed here by the hydrogen exchange method. The hydrogen exchange rates indicate that 46 amide protons, distributed throughout the structure of apoflavodoxin, exchange relatively slowly at pH 7.0 (k(ex) < 10(-1) min(-1)). According to their distribution in the structure, protein stability is highest on the beta-sheet, helix 4, and on the layer formed by helices 1 and 5. The exchange kinetics of Anabaena apoflavodoxin was compared with those of the apoflavodoxin from Azotobacter, with which it shares a 48% sequence identity, and with Che Y and cutinase, two other alpha/beta (21345) proteins with no significant sequence homology with flavodoxins. Both similarities and differences are observed in the cores of these proteins. It is of interest that a cluster of a few structurally equivalent residues in the central beta-strands and in helix 5 is common to the cores.     

Folding of DsbB in mixed micelles: a kinetic analysis of the stability of a bacterial membrane protein

Measuring the stability of integrated membrane proteins under equilibrium conditions is hampered by the nature of the proteins' amphiphilic environment. While intrinsic fluorescence is a useful probe for structural changes in water-soluble proteins, the fluorescence of membrane proteins is sensitive to changes in lipid and detergent composition. As an attempt to overcome this problem, I present a kinetic analysis of the folding of a membrane protein, disulfide bond reducing protein B (DsbB), in a mixed micelle system consisting of varying molar ratios of sodium dodecyl sulfate (SDS) and dodecyl maltoside (DM). This analysis incorporates both folding and unfolding rates, making it possible to determine both the stability of the native state and the process by which the protein folds. Refolding and unfolding occur on the second to millisecond timescale and involve only one relaxation phase, when monitored by conventional stopped-flow. The kinetic data indicate that denaturation occurs around 0.3 mole fraction of SDS, in agreement with CD analysis and acrylamide quenching data. The rate constants have been fit to a three-state folding scheme involving the SDS-denatured state, the native state and an unfolding intermediate that accumulates only under unfolding conditions at high mole fractions of SDS. The stability of DsbB is around 4.4 kcal/mol in DM, and this is halved upon reduction of the two periplasmic disulfide bonds, and is sensitive to mutagenesis. With the caveat that kinetic data are always open to alternative interpretations, time-resolved studies in mixed micelles provide a useful approach to measure membrane protein stability over a wide range of concentrations of SDS and DM, as well as a framework for the future characterization of the DsbB folding mechanism.     

Pathway and stability of protein folding.

We describe an experimental approach to the problem of protein folding and stability which measures interaction energies and maps structures of intermediates and transition states during the folding pathway. The strategy is based on two steps. First, protein engineering is used to remove interactions that stabilize defined positions in barnase, the RNAse from Bacillus amyloliquefaciens. The consequent changes in stability are measured from the changes in free energy of unfolding of the protein. Second, each mutation is used as a probe of the structure around the wild-type side chain during the folding process. Kinetic measurements are made on the folding and unfolding of wild-type and mutant proteins. The kinetic and thermodynamic data are combined and analysed to show the role of individual side chains in the stabilization of the folded, transition and intermediate states of the protein. The protein engineering experiments are corroborated by nuclear magnetic resonance studies of hydrogen exchange during the folding process. Folding is a multiphasic process in which alpha-helices and beta-sheet are formed relatively early. Formation of the hydrophobic core by docking helix and sheet is (partly) rate determining. The final steps involve the forming of loops and the capping of the N-termini of helices.     

The structure of a two-disulfide intermediate assists in elucidating the oxidative folding pathway of a cyclic cystine knot protein

We have determined the three-dimensional structure of a two-disulfide intermediate (Cys(8)-Cys(20), Cys(14)-Cys(26)) on the oxidative folding pathway of the cyclotide MCoTI-II. Cyclotides have a range of bioactivities and, because of their exceptional stability, have been proposed as potential molecular scaffolds for drug design applications. The three-dimensional structure of the stable two-disulfide intermediate shows for the most part identical secondary and tertiary structure to the native state. The only exception is a flexible loop, which is collapsed onto the protein core in the native state, whereas in the intermediate it is more loosely associated with the remainder of the protein. The results suggest that the native fold of the peptide does not represent the free energy minimum in the absence of the Cys(1)-Cys(18) disulfide bridge and that although there is not a large energy barrier, the peptide must transiently adopt an energetically unfavorable state before the final disulfide can form.