Folding pathway of FKBP12 and characterisation of the transition state.

The folding pathway of human FKBP12, a 12 kDa FK506-binding protein (immunophilin), has been characterised. Unfolding and refolding rate constants have been determined over a wide range of denaturant concentrations and data are shown to fit to a two-state model of folding in which only the denatured and native states are significantly populated, even in the absence of denaturant. This simple model for folding, in which no intermediate states are significantly populated, is further supported from stopped-flow circular dichroism experiments in which no fast "burst" phases are observed. FKBP12, with 107 residues, is the largest protein to date which folds with simple two-state kinetics in water (kF=4 s(-1)at 25 degrees C). The topological crossing of two loops in FKBP12, a structural element suggested to cause kinetic traps during folding, seems to have little effect on the folding pathway.The transition state for folding has been characterised by a series of experiments on wild-type FKBP12. Information on the thermodynamic nature of, the solvent accessibility of, and secondary structure in, the transition state was obtained from experiments measuring the unfolding and refolding rate constants as a function of temperature, denaturant concentration and trifluoroethanol concentration. In addition, unfolding and refolding studies in the presence of ligand provided information on the structure of the ligand-binding pocket in the transition state. The data suggest a compact transition state relative to the unfolded state with some 70 % of the surface area buried. The ligand-binding site, which is formed mainly by two loops, is largely unstructured in the transition state. The trifluoroethanol experiments suggest that the alpha-helix may be formed in the transition state. These results are compared with results from protein engineering studies and molecular dynamics simulations (see the accompanying paper).     

Time-resolved small-angle X-ray scattering study of the folding dynamics of barnase

Structural changes of barnase during folding were investigated using time-resolved small-angle X-ray scattering (SAXS). The folding of barnase involves a burst-phase intermediate, sometimes designated as the denatured state under physiological conditions, D_phys, and a second hidden intermediate. Equilibrium SAXS measurements showed that the radius of gyration (R_g) of the guanidine unfolded state (U) is 26.9 ± 0.7 Å, which remains largely constant over a wide denaturant concentration range. Time-resolved SAXS measurements showed that the R_g value extrapolated from kinetic R_g data to time zero, R_g,0, is 24.3 ± 0.1 Å, which is smaller than that of U but which is expanded from that of folding intermediates of other proteins with similar chain lengths (19 Å). After the burst-phase change, a single-exponential reduction in R_g² was observed, which corresponds to the formation of the native state for the major component containing the native trans proline isomer. We estimated R_g of the minor component of D_phys containing the non-native cis proline isomer (D_phys,cis) to be 25.7 ± 0.6 Å. Moreover, R_g of the major component of D_phys containing the native proline isomer (D_phys,tra) was estimated as 23.9 ± 0.2 Å based on R_g,0. Consequently, both components of the burst-phase intermediate of barnase (D_phys,tra and D_phys,cis) are still largely expanded. It was inferred that D_phys possesses the N-terminal helix and the center of the β-sheet formed independently and that the formation of the remainder of the protein occurs in the slower phase.     

Folding nuclei in proteins

When a protein folds or unfolds, it passes through many half-folded microstates. Only a few of them can accumulate and be seen experimentally, and this happens only when the folding (or unfolding) occurs far from the point of thermodynamic equilibrium between the native and denatured states. The universal features of folding, though, are observed just close to the equilibrium point. Here the 'two-state' transition proceeds without any accumulation of metastable intermediates, and only the transition state ('folding nucleus') is outlined by its key influence on the folding-unfolding kinetics. Our aim is to review recent experimental and theoretical studies of the folding nuclei.     

Folding Nuclei in Proteins

When a protein folds or unfolds, it passes through many half-folded microstates. Only a few of them can accumulate and be seen experimentally, and this happens only when the folding (or unfolding) occurs far from the point of thermodynamic equilibrium between the native and denatured states. The universal features of folding, though, are observed in the vicinity of the equilibrium point. Here the two-state transition proceeds without any accumulation of metastable intermediates, and only the transition state (folding nucleus) is outlined by its key influence on the folding/unfolding kinetics. This review covers recent experimental and theoretical studies of folding nuclei.     

Folding of a designed simple ankyrin repeat protein

Ankyrin repeats (AR) are 33-residue motifs containing a beta-turn, followed by two alpha-helices connected by a loop. AR occur in tandem arrangements and stack side-by-side to form elongated domains involved in very different cellular tasks. Recently, consensus libraries of AR repeats were constructed. Protein E1_5 represents a member of the shortest library, and consists of only a single consensus repeat flanked by designed N- and C-terminal capping repeats. Here we present a biophysical characterization of this AR domain. The protein is compactly folded, as judged from the heat capacity of the native state and from the specific unfolding enthalpy and entropy. From spectroscopic data, thermal and urea-induced unfolding can be modeled by a two-state transition. However, scanning calorimetry experiments reveal a deviation from the two-state behavior at elevated temperatures. Folding and unfolding at 5 degrees C both follow monoexponential kinetics with k(folding) = 28 sec(-1) and k(unfolding) = 0.9 sec(-1). Kinetic and equilibrium unfolding parameters at 5 degrees C agree very well. We conclude that E1_5 folds in a simple two-state manner at low temperatures while equilibrium intermediates become populated at higher temperatures. A chevron-plot analysis indicates that the protein traverses a very compact transition state along the folding/unfolding pathway. This work demonstrates that a designed minimal ankyrin repeat protein has the thermodynamic and kinetic properties of a compactly folded protein, and explains the favorable properties of the consensus framework.     

Is protein unfolding the reverse of protein folding? A lattice simulation analysis.

Simulations and experiments that monitor protein unfolding under denaturing conditions are commonly employed to study the mechanism by which a protein folds to its native state in a physiological environment. Due to the differences in conditions and the complexity of the reaction, unfolding is not necessarily the reverse of folding. To assess the relevance of temperature initiated unfolding studies to the folding problem, we compare the folding and unfolding of a 125-residue protein model by Monte Carlo dynamics at two temperatures; the lower one corresponds to the range used in T -jump experiments and the higher one to the range used in unfolding simulations of all-atom models. The trajectories that lead from the native state to the denatured state at these elevated temperatures are less diverse than those observed in the folding simulations. At the lower temperature, the system unfolds through a mandatory intermediate that corresponds to a local free energy minimum. At the higher temperature, no such intermediate is observed, but a similar pathway is followed. The structures contributing to the unfolding pathways resemble most closely those that make up the "fast track" of folding. The transition state for unfolding at the lower temperature (above Tm) is determined and is found to be more structured than the transition state for folding below the melting temperature. This shift towards the native state is consistent with the Hammond postulate. The implications for unfolding simulations of higher resolution models and for unfolding experiments of proteins are discussed.     

Folding of chymotrypsin inhibitor 2. 2. Influence of proline isomerization on the folding kinetics and thermodynamic characterization of the transition state of folding

The refolding of chymotrypsin inhibitor 2 (CI2) is, at least, a triphasic process. The rate constants are 53 s-1 for the major phase (77% of the total amplitude) and 0.43 and 0.024 s-1 for the slower phases (23% of the total amplitude) at 25 degrees C and pH 6.3. The multiphase nature of the refolding reaction results from heterogeneity in the denatured state because of proline isomerization. The fast phase corresponds to the refolding of the fraction of protein that has all its prolines in a native trans conformation in the denatured state. It is not catalyzed by peptidyl-prolyl isomerase. The rate-limiting step of folding for the slower phases, however, is proline isomerization, and they are both catalyzed by peptidyl-prolyl isomerase. The slowest phase has properties consistent with a process involving proline isomerization in a denatured state. In particular, the activation enthalpy is large, 16 kcal mol-1 K-1, and the rate is independent of guanidinium chloride concentration ([GdnHCl]). In comparison, the intermediate phase shows properties consistent with a process involving proline isomerization in a partially structured state. The activation enthalpy is small, 8 kcal mol-1 K-1, and the rate has a strong dependence on [GdnHCl]. Temperature dependences of the rate constants for unfolding and for the fast refolding phase, both in the absence and in the presence of GdnHCl, were used to characterize the thermodynamic nature of the transition state and its relative exposure to solvent. The Eyring plot for unfolding is linear, indicating that there is relatively little change in heat capacity between native state and transition state.(ABSTRACT TRUNCATED AT 250 WORDS)     

Folding thermodynamics and kinetics of the leucine-rich repeat domain of the virulence factor Internalin B

Although the folding of alpha-helical repeat proteins has been well characterized, much less is known about the folding of repeat proteins containing beta-sheets. Here we investigate the folding thermodynamics and kinetics of the leucine-rich repeat (LRR) domain of Internalin B (InlB), an extracellular virulence factor from the bacterium Lysteria monocytogenes. This domain contains seven tandem leucine-rich repeats, of which each contribute a single beta-strand that forms a continuous beta-sheet with neighboring repeats, and an N-terminal alpha-helical capping motif. Despite its modular structure, InlB folds in an equilibrium two-state manner, as reflected by the identical thermodynamic parameters obtained by monitoring its sigmoidal urea-induced unfolding transition by different spectroscopic probes. Although equilibrium two-state folding is common in alpha-helical repeat proteins, to date, InlB is the only beta-sheet-containing repeat protein for which this behavior is observed. Surprisingly, unlike other repeat proteins exhibiting equilibrium two-state folding, InlB also folds by a simple two-state kinetic mechanism lacking intermediates, aside from the effects of prolyl isomerization on the denatured state. However, like other repeat proteins, InlB also folds significantly more slowly than expected from contact order. When plotted against urea, the rate constants for the fast refolding and single unfolding phases constitute a linear chevron that, when fitted with a kinetic two-state model, yields thermodynamic parameters matching those observed for equilibrium folding. Based on these kinetic parameters, the transition state is estimated to comprise 40% of the total surface area buried upon folding, indicating that a large fraction of the native contacts are formed in the rate-limiting step to folding.     

The denatured state of Engrailed Homeodomain under denaturing and native conditions

Protein folding starts from the elusive form of the denatured state that is present under conditions that favour the native state. We have studied the denatured state of Engrailed Homeodomain (En-HD) under mildly and strongly denaturing conditions at the level of individual residues by NMR and more globally by conventional spectroscopy and solution X-ray scattering. We have compared these states with a destabilized mutant, L16A, which is predominantly denatured under conditions where the wild-type is native. This engineered denatured state, which could be directly studied under native conditions, was in genuine equilibrium with the native state, which could be observably populated by changing the conditions or introducing a stabilizing mutation. The denatured state had extensive native secondary structure and was significantly compact and globular. But, the side-chains and backbone were highly mobile. Non-cooperative melting of the residual structure on the denatured state of En-HD was observed, both at the residue and the molecular level, with increasingly denaturing conditions. The absence of a co-operative transition could result from the denatured state ensemble progressing through a series of intermediates or from a more general slide (second-order transition) from the compact form under native conditions to the more extended at highly denaturing conditions. In either case, the starting point for folding under native conditions is highly structured and already poised to adopt the native structure.     

The folding pathway of ubiquitin from all-atom molecular dynamics simulations

The folding (unfolding) pathway of ubiquitin is probed using all-atom molecular dynamics simulations. We dissect the folding pathway using two techniques: first, we probe the folding pathway of ubiquitin by calculating the evolution of structural properties over time and second, we identify the rate determining transition state for folding. The structural properties that we look at are hydrophobic solvent accessible surface area (SASA) and Calpha-root-mean-square deviation (rmsd). These properties on their own tell us relatively little about the folding pathway of ubiquitin; however, when plotted against each other, they become powerful tools for dissecting ubiquitin's folding mechanism. Plots of Calpha-rmsd against SASA serve as a phase space trajectories for the folding of ubiquitin. In this study, these plots show that ubiquitin folds to the native state via the population of an intermediate state. This is shown by an initial hydrophobic collapse phase followed by a second phase of secondary structure arrangement. Analysis of the structure of the intermediate state shows that it is a collapsed species with very little secondary structure. In reconciling these observations with recent experimental data, the transition that we observe in our simulations from the unfolded state (U) to the intermediate state (I) most likely occurs in the dead-time of the stopped flow instrument. The folding pathway of ubiquitin is probed further by identification of the rate-determining transition state for folding. The method used for this is essential dynamics, which utilizes a principal component analysis (PCA) on the atomic fluctuations throughout the simulation. The five transition state structures identified in silico are in good agreement with the experimentally determined transition state. The calculation of phi-values from the structures generated in the simulations is also carried out and it shows a good correlation with the experimentally measured values. An initial analysis of the denatured state shows that it is compact with fluctuating regions of nonnative secondary structure. It is found that the compactness in the denatured state is due to the burial of some hydrophobic residues. We conclude by looking at a correlation between folding kinetics and residual structure in the denatured state. A hierarchical folding mechanism is then proposed for ubiquitin.     

A Comprehensive Multidimensional-Embedded, One-Dimensional Reaction Coordinate for Protein Unfolding/Folding

The goal of the Dynameomics project is to perform, store, and analyze molecular dynamics simulations of representative proteins, of all known globular folds, in their native state and along their unfolding pathways. To analyze unfolding simulations, the location of the protein along the unfolding reaction coordinate (RXN) must be determined. Properties such as the fraction of native contacts and radius of gyration are often used; however, there is an issue regarding degeneracy with these properties, as native and nonnative species can overlap. Here, we used 15 physical properties of the protein to construct a multidimensional-embedded, one-dimensional RXN coordinate that faithfully captures the complex nature of unfolding. The unfolding RXN coordinates for 188 proteins (1534 simulations and 22.9 μs in explicit water) were calculated. Native, transition, intermediate, and denatured states were readily identified with the use of this RXN coordinate. A global native ensemble based on the native-state properties of the 188 proteins was created. This ensemble was shown to be effective for calculating RXN coordinates for folds outside the initial 188 targets. These RXN coordinates enable, high-throughput assignment of conformational states, which represents an important step in comparing protein properties across fold space as well as characterizing the unfolding of individual proteins.     

Denatured state is critical in determining the properties of model proteins designed on different folds

The thermodynamics of proteins designed on three common folds (SH3, chymotrypsin inhibitor 2 [CI2], and protein G) is studied with a simplified C(alpha) model and compared with the thermodynamics of proteins designed on random-generated folds. The model allows to design sequences to fold within a dRMSD ranging from 1.2 to 4.2 A from the crystallographic native conformation and to study properties that are hard to be measured experimentally. It is found that the denatured state of all of them is not random but is, to different extents, partially structured. The degree of structure is more abundant for SH3 and protein G, giving rise to a weaker stability but a more efficient folding kinetics than CI2 and, even more, than the random-generated folds. Consequently, the features of the unfolded state seem to be as important in the determination of the thermodynamic properties of these proteins as the features of the native state.     

Folding/Unfolding Kinetics of Apomyoglobin

The equilibrium and kinetic folding/unfolding of apomyoglobin (ApoMb) were studied at pH 6.2, 11 °C by recording tryptophan fluorescence. The equilibrium unfolding of ApoMb in the presence of urea was shown to involve accumulation of an intermediate state, which had a higher fluorescence intensity as compared with the native and unfolded states. The folding proceeded through two kinetic phases, a rapid transition from the unfolded to the intermediate state and a slow transition from the intermediate to the native state. The accumulation of the kinetic intermediate state was observed in a wide range of urea concentrations. The intermediate was detected even in the region corresponding to the unfolding limb of the chevron plot. Urea concentration dependence was obtained for the observed folding/unfolding rate. The shape of the dependence was compared with that of two-state proteins characterized by a direct transition from the unfolded to the native state.     

Understanding the Mechanism of Prosegment-catalyzed Folding by Solution NMR Spectroscopy

Multidomain protein folding is often more complex than a two-state process, which leads to the spontaneous folding of the native state. Pepsin, a zymogen-derived enzyme, without its prosegment (PS), is irreversibly denatured and folds to a thermodynamically stable, non-native conformation, termed refolded pepsin, which is separated from native pepsin by a large activation barrier. While it is known that PS binds refolded pepsin and catalyzes its conversion to the native form, little structural details are known regarding this conversion. In this study, solution NMR was used to elucidate the PS-catalyzed folding mechanism by examining the key equilibrium states, e.g. native and refolded pepsin, both in the free and PS-bound states, and pepsinogen, the zymogen form of pepsin. Refolded pepsin was found to be partially structured and lacked the correct domain-domain structure and active-site cleft formed in the native state. Analysis of chemical shift data revealed that upon PS binding refolded pepsin folds into a state more similar to that of pepsinogen than to native pepsin. Comparison of pepsin folding by wild-type and mutant PSs, including a double mutant PS, indicated that hydrophobic interactions between residues of prosegment and refolded pepsin lower the folding activation barrier. A mechanism is proposed for the binding of PS to refolded pepsin and how the formation of the native structure is mediated.     

Conformational detours during folding of a collapsed state

The protein S6 is a useful model to probe the role of partially folded states in the folding process. In the absence of salt, S6 folds from the denatured state D to the native state N without detectable intermediates. High concentrations of sodium sulfate induce the accumulation of a collapsed state C, which is off the direct folding route. However, the mutation VA85 enables S6 to fold from C directly to N through the transition state TS(C). According to the denaturant dependence of this reaction, TS(C) and C are equally compact, but the data are difficult to deconvolute. Therefore, I have measured the heat capacities (DeltaC(p)) for the D-->C and C-->TS(C) transitions. The DeltaC(p)-values suggest that C needs to increase its surface area in order to fold directly to N. This underlines that it is a misfolded state that can only fold by at least partial unfolding. In contrast to the C-state formed by S6 wildtype, the VA85 C-state is just as compact as the native state, and this may be a prerequisite for direct folding. Individual "gatekeeper" residues may thus play a disproportionately large role in guiding proteins through different folding pathways.     

The notch ankyrin domain folds via a discrete, centralized pathway

The Notch ankyrin repeat domain contains seven ankyrin sequence repeats, six of which adopt very similar structures. To determine if folding proceeds along parallel pathways and the order in which repeats become structured during folding, we examined the effect of analogous destabilizing Ala-->Gly substitutions in each repeat on folding kinetics. We find that folding proceeds to an on-pathway kinetic intermediate through a transition state ensemble containing structure in repeats three through five. Repeats two, six, and seven remain largely unstructured in this intermediate, becoming structured in a second kinetic step that leads to the native state. These data suggest that the Notch ankyrin domain folds according to a discrete kinetic pathway despite structural redundancy in the native state and highlight the importance of sequence-specific interactions in controlling pathway selection. This centralized pathway roughly corresponds to a low energy channel through the experimentally determined energy landscape.     

Folding of dimeric methionine adenosyltransferase III: identification of two folding intermediates

Methionine adenosyl transferase (MAT) is an essential enzyme that synthesizes AdoMet. The liver-specific MAT isoform, MAT III, is a homodimer of a 43.7-kDa subunit that organizes in three nonsequential alpha-beta domains. Although MAT III structure has been recently resolved, little is known about its folding mechanism. Equilibrium unfolding and refolding of MAT III, and the monomeric mutant R265H, have been monitored using different physical parameters. Tryptophanyl fluorescence showed a three-state folding mechanism. The first unfolding step was a folding/association process as indicated by its dependence on protein concentration. The monomeric folding intermediate produced was the predominant species between 1.5 and 3 m urea. It had a relatively compact conformation with tryptophan residues and hydrophobic surfaces occluded from the solvent, although its N-terminal region may be very unstructured. The second unfolding step monitored the denaturation of the intermediate. Refolding of the intermediate showed first order kinetics, indicating the presence of a kinetic intermediate within the folding/association transition. Its presence was confirmed by measuring the 1,8-anilinonaphtalene-8-sulfonic acid binding in the presence of tripolyphosphate. We propose that the folding rate-limiting step is the formation of an intermediate, probably a structured monomer with exposed hydrophobic surfaces, that rapidly associates to form dimeric MAT III.     

Mechanisms of the multiphasic kinetics in the folding and unfolding of globular proteins

The multiphasic kinetics of the protein folding and unfolding processes are examined for a “cluster model” with only two thermodynamically stable macroscopic states, native (N) and denatured (D), which are essentially distributions of microscopic states. The simplest kinetic schemes consistent with the model are: N-(fast) → I-(slow) → D for unfolding and N ← (fast)-D₂ ← (slow)-D₁ for refolding. The fast phase during the unfolding process can be visualized as the redistribution of the native population N to I within its free energy valley. Then, this population crosses over the free energy barrier to the denatured state D in the slow phase. Therefore, the macrostate I is a kinetic intermediate which is not stable at equilibrium. For the refolding process, the initial equilibrium distribution of the denatured state D appears to be separated into D₁ and D₂ in the final condition because of the change in position of the free energy barrier. The fast refolding species D₂ is due to the “leak” from the broadly distributed D state, while the rest is the slow refolding species D₁, which must overpass the free energy barrier to reach N. At an early stage of the folding process the amino acid chain is considered to be composed of several locally ordered regions, which we call clusters, connected by random coil chain parts. Thus, the denatured state contains different sizes and distributions of clusters depending on the external condition. A later stage of the folding process is the association of smaller clusters. The native state is expressed by a maximum-size cluster with possible fluctuation sites reflecting this association. A general discussion is given of the correlation between the kinetics and thermodynamics of proteins from the overall shape of the free energy function. The cluster model provides a conceptual link between the folding kinetics and the structural patterns of globular proteins derived from the X-ray crystallographic data.     

The structure of the major transition state for folding of an FF domain from experiment and simulation

We have analysed the transition state of folding of the four-helix FF domain from HYPA/FBP11 by high-resolution experiment and simulation as part of a continuing effort to understand the principles of folding and the refinement of predictive methods. The major transition state for folding was subjected to a Phi-value analysis utilising 50 mutants. The transition state contained a nucleus for folding centred around the end of helix 1 (H1) and the beginning of helix 2 (H2). Secondary structure in this region was fully formed (PhiF=0.9-1) and tertiary interactions were well developed. Interactions in the distal part of the native structure were weak (PhiF=0-0.2). The hydrophobic core and other parts of the protein displayed intermediate Phi-values, suggesting that interactions coalesce as the end of H1 and beginning of H2 are in the process of being formed. The distribution of Phi-values resembled that of barnase, which folds via an intermediate, rather than that of CI2 which folds by a concerted nucleation-condensation mechanism. The overall picture of the transition state structure identified in molecular dynamics simulations is in qualitative agreement, with the turn connecting H1 and H2 being formed, a loosened core, and H4 partially unfolded and detached from the core. There are some differences in the details and interpretation of specific Phi-values.