Influence of denatured and intermediate states of folding on protein aggregation

We simulate the aggregation thermodynamics and kinetics of proteins L and G, each of which self-assembles to the same alpha/beta [corrected] topology through distinct folding mechanisms. We find that the aggregation kinetics of both proteins at an experimentally relevant concentration exhibit both fast and slow aggregation pathways, although a greater proportion of protein G aggregation events are slow relative to those of found for protein L. These kinetic differences are correlated with the amount and distribution of intrachain contacts formed in the denatured state ensemble (DSE), or an intermediate state ensemble (ISE) if it exists, as well as the folding timescales of the two proteins. Protein G aggregates more slowly than protein L due to its rapidly formed folding intermediate, which exhibits native intrachain contacts spread across the protein, suggesting that certain early folding intermediates may be selected for by evolution due to their protective role against unwanted aggregation. Protein L shows only localized native structure in the DSE with timescales of folding that are commensurate with the aggregation timescale, leaving it vulnerable to domain swapping or nonnative interactions with other chains that increase the aggregation rate. Folding experiments that characterize the structural signatures of the DSE, ISE, or the transition state ensemble (TSE) under nonaggregating conditions should be able to predict regions where interchain contacts will be made in the aggregate, and to predict slower aggregation rates for proteins with contacts that are dispersed across the fold. Since proteins L and G can both form amyloid fibrils, this work also provides mechanistic and structural insight into the formation of prefibrillar species.     

Gatekeepers in the ribosomal protein s6: thermodynamics, kinetics, and folding pathways revealed by a minimalist protein model

We investigate the effect of structural gatekeepers on the folding of the ribosomal protein S6. Folding thermodynamics and early refolding kinetics are studied for this system utilizing computer simulations of a minimalist protein model. When gatekeepers are eliminated, the thermodynamic signature of a folding intermediate emerges, and a marked decrease in folding efficiency is observed. We explain the prerequisites that determine the "strength" of a given gatekeeper. The investigated gatekeepers are found to have distinct functions, and to guide the folding and time-dependent packing of non-overlapping secondary structure elements in the protein. Gatekeepers avoid kinetic traps during folding by favoring the formation of "productive topologies" on the way to the native state. The trends in folding rates in the presence/absence of gatekeepers observed for our minimalist model of S6 are in very good agreement with experimental data on this protein.     

The "first in-last out" hypothesis on protein folding revisited

We calculated profiles for mean residue depth, contact order, and number of contacts in the native structure of a series of proteins for which folding has been studied extensively, the chymotrypsin inhibitor 2, the SH3 module from the src tyrosine kinase, the small ribonuclease barnase, the bacterial immunity protein Im7, and apomyoglobin. We compared these profiles with experimental data from equilibrium or pulse labeling hydrogen-deuterium exchange obtained from NMR and phi values obtained from the protein engineering approach. We find a good qualitative agreement between the hierarchy of formation of topological elements during the folding process and the ranking of secondary structure elements in terms of residue depth. Residues that are most deeply buried in the core of the native protein usually belong to stretches of secondary structure elements that are formed early in the folding pathway. Residue depth can thus provide a useful and simple tool for the design of folding experiments.     

Toward minimalist models of larger proteins: a ubiquitin-like protein

Our recently developed off-lattice bead model capable of simulating protein structures with mixed alpha/beta content has been extended to model the folding of a ubiquitin-like protein and provides a means for examining the more complex kinetics involved in the folding of larger proteins. Using trajectories generated from constant-temperature Langevin dynamics simulations and sampling with the multiple multi-histogram method over five-order parameters, we are able to characterize the free energy landscape for folding and find evidence for folding through compact intermediates. Our model reproduces the observation that the C-terminus loop structure in ubiquitin is the last to fold in the folding process and most likely plays a spectator role in the folding kinetics. The possibility of a productive metastable intermediate along the folding pathway consisting of collapsed states with no secondary structure, and of intermediates or transition structures involving secondary structural elements occurring early in the sequence, is also supported by our model. The kinetics of folding remain multi-exponential below the folding temperature, with glass-like kinetics appearing at T/T(f) approximately 0.86. This new physicochemical model, designed to be predictive, helps validate the value of modeling protein folding at this level of detail for genomic-scale studies, and motivates further studies of other protein topologies and the impact of more complex energy functions, such as the addition of solvation forces.     

Matching simulation and experiment: a new simplified model for simulating protein folding.

Simulations of simplified protein folding models have provided much insight into solving the protein folding problem. We propose here a new off-lattice bead model, capable of simulating several different fold classes of small proteins. We present the sequence for an alpha/beta protein resembling the IgG-binding proteins L and G. The thermodynamics of the folding process for this model are characterized using the multiple multihistogram method combined with constant-temperature Langevin simulations. The folding is shown to be highly cooperative, with chain collapse nearly accompanying folding. Two parallel folding pathways are shown to exist on the folding free energy landscape. One pathway contains an intermediate--similar to experiments on protein G, and one pathway contains no intermediates-similar to experiments on protein L. The folding kinetics are characterized by tabulating mean-first passage times, and we show that the onset of glasslike kinetics occurs at much lower temperatures than the folding temperature. This model is expected to be useful in many future contexts: investigating questions of the role of local versus nonlocal interactions in various fold classes, addressing the effect of sequence mutations affecting secondary structure propensities, and providing a computationally feasible model for studying the role of solvation forces in protein folding.     

Secondary structure length as a determinant of folding rate of proteins with two- and three-state kinetics

We present a simple method for determining the folding rates of two- and three-state proteins from the number of residues in their secondary structures (secondary structure length). The method is based on the hypothesis that two- and three-state foldings share a common pattern. Three-state proteins first condense into metastable intermediates, subsequent forming of alpha-helices, turns, and beta-sheets at slow rate-limiting step. The folding rate of such proteins anticorrelate with the length of these beta-secondary structures. It is also assumed that in two-state folding, rapidly folded alpha-helices and turns may facilitate formation of fleeting unobservable intermediates and thus show two-state behavior. There is an inverse relationship between the folding rate and the length of beta-sheets and loops. Our study achieves 94.0 and 88.1% correlations with folding rates determined experimentally for 21 three- and 38 two-state proteins, respectively, suggesting that protein-folding rates are determined by the secondary structure length. The kinetic kinds are selected on the basis of a competitive formation of hydrophobic collapse and alpha-structure in early intermediates.     

Models for protein folding and nature's choice of protein as catalyst

The study of protein folding and unfolding pathways lends a fascinating dimension to protein biochemistry. Several models for protein folding have been postulated. Two powerful probes used in protein folding study are far UV-CD monitored stopped flow kinetics and pulse hydrogen exchange in conjunction with NMR. The formation of molten globule, which is an intermediate possessing secondary structure but not a well packed tertiary structure, is now emerging as a common feature on the folding pathway of many proteins. The molten globule is recognized by a class of molecules called chaperones which act as accelerators of protein folding. This article ends by elucidating why proteins are Nature's choice as catalysts.     

Identification and characterization of key substructures involved in the early folding events of a (beta/alpha)8-barrel protein as studied by experimental and computational methods

A number of studies have examined the structural properties of late folding intermediates of (beta/alpha)8-barrel proteins involved in tryptophan biosynthesis, whereas there is little information available about the early folding events of these proteins. To identify the contiguous polypeptide segments important to the folding of the (beta/alpha)8-barrel protein Escherichia coli N-(5'-phosphoribosyl)anthranilate isomerase, we structurally characterized fragments and circularly permuted forms of the protein. We also simulated thermal unfolding of the protein using molecular dynamics. Our fragmentation experiments demonstrate that the isolated (beta/alpha)(1-4)beta5 fragment is almost as stable as the full-length protein. The far and near-UV CD spectra of this fragment are indicative of native-like secondary and tertiary structures. Structural analysis of the circularly permutated proteins shows that if the protein is cleaved within the two N-terminal betaalpha modules, the amount of secondary structure is unaffected, whereas, when cleaved within the central (beta/alpha)(3-4)beta5 segment, the protein simply cannot fold. An ensemble of the denatured structures produced by thermal unfolding simulations contains a persistent local structure comprised of beta3, beta4 and beta5. The presence of this three-stranded beta-barrel suggests that it may be an important early-stage folding intermediate. Interactions found in (beta/alpha)(3-4)beta5 may be essential for the early events of ePRAI folding if they provide a nucleation site that directs folding.     

Effects of the difference in the unfolded-state ensemble on the folding of Escherichia coli dihydrofolate reductase

The unfolded state of a protein is an ensemble of a large number of conformations ranging from fully extended to compact structures. To investigate the effects of the difference in the unfolded-state ensemble on protein folding, we have studied the structure, stability, and folding of "circular" dihydrofolate reductase (DHFR) from Escherichia coli in which the N and C-terminal regions are cross-linked by a disulfide bond, and compared the results with those of disulfide-reduced "linear" DHFR. Equilibrium studies by circular dichroism, difference absorption spectra, solution X-ray scattering, and size-exclusion chromatography show that whereas the native structures of both proteins are essentially the same, the unfolded state of circular DHFR adopts more compact conformations than the unfolded state of the linear form, even with the absence of secondary structure. Circular DHFR is more stable than linear DHFR, which may be due to the decrease in the conformational entropy of the unfolded state as a result of circularization. Kinetic refolding measurements by stopped-flow circular dichroism and fluorescence show that under the native conditions both proteins accumulate a burst-phase intermediate having the same structures and both fold by the same complex folding mechanism with the same folding rates. Thus, the effects of the difference in the unfolded state of circular and linear DHFRs on the refolding reaction are not observed after the formation of the intermediate. This suggests that for the proteins with close termini in the native structure, early compaction of a protein molecule to form a specific folding intermediate with the N and C-terminal regions in close proximity is a crucial event in folding. If there is an enhancement in the folding reflecting the reduction in the breadth of the unfolded-state ensemble for circular DHFR, this acceleration must occur in the sub-millisecond time-range.     

Folding studies on a knotted protein

YibK is a 160 residue homodimeric protein belonging to the SPOUT class of methyltransferases. Proteins in this group all display a unique topological feature; the backbone polypeptide chain folds to form a deep trefoil knot. Such knotted structures were completely unpredicted, it being thought impossible for a protein to fold efficiently in this way. However, they are becoming more common and there are now a growing number of examples in the Protein Data Bank. These intriguing knotted structures represent a new and significant challenge in the field of protein folding. Here, we present an initial characterisation of the folding of YibK, one of the smallest knotted proteins to be identified. This is the first detailed folding study on a knotted protein to be reported. We have established conditions under which the protein can be denatured reversibly in vitro using urea, thereby showing that molecular chaperones are not required for the efficient folding of this protein. A series of equilibrium unfolding experiments were performed over a 400-fold range of protein concentration. Both secondary and tertiary structural probes show a single, protein concentration-dependent unfolding transition, and data are most consistent with a three-state equilibrium denaturation model involving a monomeric intermediate. Thermodynamic parameters obtained from the fit of the data to this model indicate that the intermediate is a stable species with appreciable secondary and tertiary structure; whether the topological knot remains in the intermediate state is still to be shown. Together, these results demonstrate that, despite its complex knotted structure, YibK is able to fold efficiently and behaves remarkably similarly to other dimeric proteins under equilibrium conditions.     

The hydrogen exchange core and protein folding.

A database of hydrogen-deuterium exchange results has been compiled for proteins for which there are published rates of out-exchange in the native state, protection against exchange during folding, and out-exchange in partially folded forms. The question of whether the slow exchange core is the folding core (Woodward C, 1993, Trends Biochem Sci 18:359-360) is reexamined in a detailed comparison of the specific amide protons (NHs) and the elements of secondary structure on which they are located. For each pulsed exchange or competition experiment, probe NHs are shown explicitly; the large number and broad distribution of probe NHs support the validity of comparing out-exchange with pulsed-exchange/competition experiments. There is a strong tendency for the same elements of secondary structure to carry NHs most protected in the native state, NHs first protected during folding, and NHs most protected in partially folded species. There is not a one-to-one correspondence of individual NHs. Proteins for which there are published data for native state out-exchange and theta values are also reviewed. The elements of secondary structure containing the slowest exchanging NHs in native proteins tend to contain side chains with high theta values or be connected to a turn/loop with high theta values. A definition for a protein core is proposed, and the implications for protein folding are discussed. Apparently, during folding and in the native state, nonlocal interactions between core sequences are favored more than other possible nonlocal interactions. Other studies of partially folded bovine pancreatic trypsin inhibitor (Barbar E, Barany G, Woodward C, 1995, Biochemistry 34:11423-11434; Barber E, Hare M, Daragan V, Barany G, Woodward C, 1998, Biochemistry 37:7822-7833), suggest that developing cores have site-specific energy barriers between microstates, one disordered, and the other(s) more ordered.     

An Overlapping Region between the Two Terminal Folding Units of the Outer Surface Protein A (OspA) Controls Its Folding Behavior

Although many naturally occurring proteins consist of multiple domains, most studies on protein folding to date deal with single-domain proteins or isolated domains of multi-domain proteins. Studies of multi-domain protein folding are required for further advancing our understanding of protein folding mechanisms. Borrelia outer surface protein A (OspA) is a β-rich two-domain protein, in which two globular domains are connected by a rigid and stable single-layer β-sheet. Thus, OspA is particularly suited as a model system for studying the interplays of domains in protein folding. Here, we studied the equilibria and kinetics of the urea-induced folding–unfolding reactions of OspA probed with tryptophan fluorescence and ultraviolet circular dichroism. Global analysis of the experimental data revealed compelling lines of evidence for accumulation of an on-pathway intermediate during kinetic refolding and for the identity between the kinetic intermediate and a previously described equilibrium unfolding intermediate. The results suggest that the intermediate has the fully native structure in the N-terminal domain and the single layer β-sheet, with the C-terminal domain still unfolded. The observation of the productive on-pathway folding intermediate clearly indicates substantial interactions between the two domains mediated by the single-layer β-sheet. We propose that a rigid and stable intervening region between two domains creates an overlap between two folding units and can energetically couple their folding reactions.     

Evidence for identity between the equilibrium unfolding intermediate and a transient folding intermediate: a comparative study of the folding reactions of alpha-lactalbumin and lysozyme

The refolding kinetics of alpha-lactalbumin at different concentrations of guanidine hydrochloride have been investigated by means of kinetic circular dichroism and stopped-flow absorption measurements. The refolding reaction consists of at least two stages, the instantaneous accumulation of the transient intermediate that has peptide secondary structure and the subsequent slow process associated with formation of tertiary structure. The transient intermediate is compared with the well-characterized equilibrium intermediate observed during the denaturant-induced unfolding. Stabilities of the secondary structures against the denaturant, affinities for Ca2+, and tryptophan absorption properties of the transient and equilibrium intermediates were investigated. In all of these respects, the transient intermediate is identical with the equilibrium one, demonstrating the validity of the use of the equilibrium intermediate as a model of the folding intermediate. Essentially the same transient intermediate was also detected in the folding of lysozyme, the protein known to be homologous to alpha-lactalbumin but whose equilibrium unfolding is represented as a two-state reaction. The stability and cooperativity of the secondary structure of the intermediate of lysozyme are compared with those of alpha-lactalbumin. The results show that the protein folding occurring via the intermediate is not limited to the proteins that show equilibrium intermediates. Although the unfolding equilibria of most proteins are well approximated as a two-state reaction, the two-state hypothesis may not be applicable to the folding reaction under the native condition. Two models of protein folding, intermediate-controlled folding model and multiple-pathway folding model, which are different in view of the role of the intermediate in determining the pathway of folding, are also discussed.     

Testing the relationship between foldability and the early folding events of dihydrofolate reductase from Escherichia coli

A "folding element" is a contiguous peptide segment crucial for a protein to be foldable and is a new concept that could assist in our understanding of the protein-folding problem. It is known that the presence of the complete set of folding elements of dihydrofolate reductase (DHFR) from Escherichia coli is essential for the protein to be foldable. Since almost all of the amino acid residues known to be involved in the early folding events of DHFR are located within the folding elements, a close relationship between the folding elements and early folding events is hypothesized. In order to test this hypothesis, we have investigated whether or not the early folding events are preserved in circular permutants and topological mutants of DHFR, in which the order of the folding elements is changed but the complete set of folding elements is present. The stopped-flow circular dichroism (CD) measurements show that the CD spectra at the early stages of folding are similar among the mutants and the wild-type DHFR, indicating that the presence of the complete set of folding elements is sufficient to preserve the early folding events. We have further examined whether or not sequence perturbation on the folding elements by a single amino acid substitution affects the early folding events of DHFR. The results show that the amino acid substitutions inside of the folding elements can affect the burst-phase CD spectra, whereas the substitutions outside do not. Taken together, these results indicate that the above hypothesis is true, suggesting a close relationship between the foldability of a protein and the early folding events. We propose that the folding elements interact with each other and coalesce to form a productive intermediate(s) early in the folding, and these early folding events are important for a protein to be foldable.     

Folding mechanisms of proteins with high sequence identity but different folds

The problem of how a protein folds from a linear chain of amino acids to the three-dimensional structure necessary for function is often investigated using proteins with a low degree of sequence identity that adopt different folds. The design of pairs of proteins with a high degree of sequence identity but different folds offers the opportunity for a complementary study; in two highly similar sequences, which residues are the most important in directing folding to a particular structure? Here we use molecular dynamics simulations to characterize the folding-unfolding pathways of a pair of proteins designed by Bryan and co-workers [Alexander, P. A., et al. (2005) Biochemistry 44, 14045-14054; He, Y. N., et al. (2005) Biochemistry 44, 14055-14061]. Despite being 59% identical, the two protein sequences fold to two different structures. The first sequence folds to the alpha+beta protein G structure and the second to the all-alpha-helical protein A structure. We show that the final protein structure is determined early along the folding pathway. In folding to the protein G structure, the single alpha-helix (alpha1) and the beta3-beta4 turn fold early. Formation of the hairpin turn essentially prevents folding to helical structure in this region of the protein. This early structure is then consolidated by formation of long-range hydrophobic interactions between alpha1 and the beta3-beta4 turn. The protein A sequence differs both in the residues that form the beta3-beta4 turn and also in many of the residues that form the early hydrophobic interactions in the protein G structure. Instead, in the protein A sequence, a more hierarchical mechanism is observed, with helices folding before many of the tertiary interactions are formed. We find that small, but critical, sequence differences determine the topology of the protein early along the folding pathway, which help to explain the process by which one fold can evolve into another.     

NMR analysis of the conformational properties of the trapped on-pathway folding intermediate of the bacterial immunity protein Im7

Previous work shows that the transiently populated, on-pathway intermediate in Im7 folding contains three of the four native alpha-helices docked around a core stabilised by native and non-native interactions. To determine the structure and dynamic properties of this species in more detail, we have used protein engineering to trap the intermediate at equilibrium and analysed the resulting proteins using NMR spectroscopy and small angle X-ray scattering. Four variants were created. In L53AI54A, two hydrophobic residues within helix III are truncated, preventing helix III from docking stably onto the developing hydrophobic core. In two other variants, the six residues encompassing the native helix III were replaced with three (H3G3) or six (H3G6) glycine residues. In the fourth variant, YY, two native tyrosine residues (Tyr55 and Tyr56) were re-introduced into H3G6 to examine their role in determining the properties of the intermediate ensemble. All four variants show variable peak intensities and broad peak widths, consistent with these proteins being conformationally dynamic. Chemical shift analyses demonstrated that L53AI54A and YY contain native-like secondary structure in helices I and IV, while helix II is partly formed and helix III is absent. Lack of NOEs and rapid NH exchange for L53AI54A, combined with detailed analysis of the backbone dynamics, indicated that the hydrophobic core of this variant is not uniquely structured, but fluctuates on the NMR timescale. The results demonstrate that though much of the native-like secondary structure of Im7 is present in the variants, their hydrophobic cores remain relatively fluid. The comparison of H3G3/H3G6 and L53AI54A/YY suggests that Tyr55 and/or Tyr56 interact with the three-helix core, leading other residues in this region of the protein to dock with the core as folding progresses. In this respect, the three-helix bundle acts as a template for formation of helix III and the creation of the native fold.     

Multi-State Proteins: Approach Allowing Experimental Determination of the Formation Order of Structure Elements in the Green Fluorescent Protein

The most complex problem in studying multi-state protein folding is the determination of the sequence of formation of protein intermediate states. A far more complex issue is to determine at what stages of protein folding its various parts (secondary structure elements) develop. The structure and properties of different intermediate states depend in particular on these parts. An experimental approach, named μ-analysis, which allows understanding the order of formation of structural elements upon folding of a multi-state protein was used in this study. In this approach the same elements of the protein secondary structure are “tested” by substitutions of single hydrophobic amino acids and by incorporation of cysteine bridges. Single substitutions of hydrophobic amino acids contribute to yielding information on the late stages of protein folding while incorporation of ss-bridges allows obtaining data on the initial stages of folding. As a result of such an μ-analysis, we have determined the order of formation of beta-hairpins upon folding of the green fluorescent protein.     

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.