Abstract: Folding and function of proteins are two aspects of proteins which are usually considered as basically unrelated phenomena that are optimized by evolution independently. From the funnel model of folding/unfolding and the associated energy landscape, we infer the paradigm that the minimum number of folding intermediates is determined by the number of all functional states of a protein ("essential" folding intermediates). Here, we demonstrate the supposed fundamental link using the Ras protein complexed with the GTP analogue GppNHp that occurs in two structural states coexisting in solution. State 2 was shown earlier to represent the effector interacting state, and the function of state 1 was hitherto unknown. By (31)P NMR spectroscopy, we demonstrate that state 1 represents the conformation interacting with guanine nucleotide exchange factors (GEFs). Denaturation experiments of the protein with a chaotropic reagent show that both functional states coexist during folding and unfolding. Application of high pressure represents another perturbation of the energy landscape, leading to an increased population of the state 1 as observed by NMR spectroscopy. The specific volume difference between the two states DeltaV(12) is 17.2 +/- 0.5 mL mol(-1), indicating that state 1 represents a more open conformation of the protein. The free energies of stabilization for state 1 and state 2 at 278 K can be determined as 8.3 and 9.8 kJ mol(-1), respectively.

Abstract: Backbone-backbone hydrogen bonds are a common feature of native protein structures, yet their thermodynamic and kinetic influence on folding has long been debated. This is reflected by the disparity between current protein folding models, which place hydrogen bond formation at different stages along the folding trajectory. For example, previous studies have suggested that the denatured state of the villin headpiece subdomain contains a residual helical structure that may provide a bias toward the folded state by confining the conformational search associated with its folding. Although helical hydrogen bonds clearly stabilize the folded state, here we show, using an amide-to-ester mutation strategy, that the formation of backbone hydrogen bonds within helices is not rate-limiting in the folding of the subdomain, thereby suggesting that such hydrogen bonds are unlikely to be formed en route from the denatured to the transition state. On the other hand, elimination of hydrogen bonds within the turn region elicits a slower folding rate, consistent with the hypothesis that these residues are involved in the formation of a folding nucleus. While illustrating a potentially conserved aspect of helix-turn-helix folding, our results further underscore the inherent importance of turns in protein supersecondary structure formation.

Abstract: We propose here KineticDB, a systematically compiled database of protein folding kinetics, which contains about 90 unique proteins. The main goal of the KineticDB is to provide users with a diverse set of protein folding rates determined experimentally. The search for determinants of protein folding is still in progress, aimed at obtaining a new understanding of the folding process. Comparison with experimental protein folding rates has been the main tool for validation of both theoretical models and empirical relationships during the last 10 years. It is, therefore, necessary to provide a researcher with as much data as possible in a simple and easy-to-use way. At present, the KineticDB contains the results of folding kinetics measurements of single-domain proteins and separate protein domains as well as short peptides without disulfide bonds. It includes data on about 90 unique proteins and many mutants that have been systematically accumulated over the last 10 years and is the largest collection of protein folding kinetic data presented as a database. The KineticDB is available at http://kineticdb.protres.ru/db/index.pl.

Abstract: Proteins possessing deeply embedded topological knots in their structure add a stimulating new challenge to the already complex protein-folding problem. The most complicated knotted topology observed to date belongs to the human enzyme ubiquitin C-terminal hydrolase UCH-L3, which is an integral part of the ubiquitin–proteasome system. The structure of UCHL3 contains five distinct crossings of its polypeptide chain, and it adopts a 52-knotted topology, making it a fascinating target for folding studies. Here, we provide the first in depth characterization of the stability and folding of UCH-L3. We show that the protein can unfold and refold reversibly in vitro without the assistance of molecular chaperones, demonstrating that all the information necessary for the protein to find its knotted native structure is encoded in the amino acid sequence, just as with any other globular protein, and that the protein does not enter into any deep kinetic traps. Under equilibrium conditions, the unfolding of UCH-L3 appears to be two-state, however, multiphasic folding and unfolding kinetics are observed and the data are consistent with a folding pathway in which two hyperfluorescent intermediates are formed. In addition, a very slow phase in the folding kinetics is shown to be limited by proline-isomerization events. Overall, the data suggest that a knotted topology, even in its most complex form, does not necessarily limit folding in vitro, however, it does seem to require a complex folding mechanism which includes the formation of several distinct intermediate species.

Abstract: Temperature-jump perturbation was used to examine the relaxation kinetics of folding of the human prion protein. Measured rates were very fast (approximately 3,000 s(-1)), with the extrapolated folding rate constant at approximately 20 degrees C in physiological conditions reaching 20,000 s(-1). By a mutational analysis of core residues, we found that only 2, on the interface of helices 2 and 3, have significant phi-values in the transition state. Interestingly, a mutation sandwiched between the above 2 residues on the helix-helix contact interface had very little effect on the overall free energy of folding but led to the formation of a monomeric misfolded state, which had to unfold to acquire the native PrP(C) conformation. Another mutation that led to a marked destabilization of the native fold also formed a misfolded intermediate, but this was aggregation-prone despite the native state of this mutant being soluble. Taken together, the data imply that this fast-folding protein has a transition state that is not compact (m value analysis gives a beta(t) value of only 0.3) but contains a developing nucleus between helices 2 and 3. The fact that a mutation in this nucleus had a negligible effect on stability but still led to formation of aberrant conformations during folding implies an easily perturbed folding mechanism. It is notable that in inherited forms of human prion disease, where point mutations produce a lethal dominant condition, 20 of the 33 amino acid replacements occur in the helix-2/3 sequence.

Abstract: Human age-onset cataracts are believed to be caused by the aggregation of partially unfolded or covalently damaged lens crystallin proteins; however, the exact molecular mechanism remains largely unknown. We have used microseconds of molecular dynamics simulations with explicit solvent to investigate the unfolding process of human lens γD-crystallin protein and its isolated domains. A partially unfolded folding intermediate of γD-crystallin is detected in simulations with its C-terminal domain (C-td) folded and N-terminal domain (N-td) unstructured, in excellent agreement with biochemical experiments. Our simulations strongly indicate that the stability and the folding mechanism of the N-td are regulated by the interdomain interactions, consistent with experimental observations. A hydrophobic folding core was identified within the C-td that is comprised of a and b strands from the Greek key motif 4, the one near the domain interface. Detailed analyses reveal a surprising non-native surface salt-bridge between Glu135 and Arg142 located at the end of the ab folded hairpin turn playing a critical role in stabilizing the folding core. On the other hand, an in silico single E135A substitution that disrupts this non-native Glu135-Arg142 salt-bridge causes significant destabilization to the folding core of the isolated C-td, which, in turn, induces unfolding of the N-td interface. These findings indicate that certain highly conserved charged residues, that is, Glu135 and Arg142, of γD-crystallin are crucial for stabilizing its hydrophobic domain interface in native conformation, and disruption of charges on the γD-crystallin surface might lead to unfolding and subsequent aggregation.

Abstract: Repeat proteins contain short, tandem arrays of simple structural motifs (20 to 40 amino acids). These stack together to form non-globular structures that are stabilised by short range interactions from residues close in primary sequence. Unlike globular proteins, they have few, if any, long range non-local stabilising interactions. One ubiquitous repeat is the tetratricopeptide motif (TPR) a 34 amino acid helix-turn-helix motif. In this article we describe the folding kinetics of a series of seven designed TPR proteins that are assembled from arraying identical designed consensus repeats (CTPRan). These range from the smallest two-repeat protein to a large ten-repeat protein ( 350 a.a). In particular, we investigate how the energy landscape changes with the addition of repeat units. The data reveal that although the CTPRa proteins have low local frustration, their highly symmetric, modular native structure is reflected in their multi-state kinetics of unfolding and folding. Moreover, although the initial folding of all CTPRan proteins involves a nucleus of similar solvent accessibility, their consequent steps to native structure change as repeat number increases. This corresponds to an increasingly complex landscape that culminates in CTPRa10 populating a misfolded, thus off-pathway, intermediate. These novel results extend our current understanding of the malleable folding pathways of repeat proteins and highlight the consequences of adding identical repeats to the energy landscape.

Abstract: The aim of this work is to shed more light on the effect of domain–domain interactions on the kinetics and the pathway of protein folding. A model protein system consisting of several single-tryptophan variants of the two-domain yeast phosphoglycerate kinase (PGK) and its individual domains was studied. Refolding was initiated from the guanidine-unfolded state by stopped-flow or manual mixing and monitored by tryptophan fluorescence from 1 msec to 1000 sec. Denaturant titrations of both individual domains showed apparent two-state unfolding transitions. Refolding kinetics of the individual domains from different denaturant concentrations, however, revealed the presence of intermediate structures during titration for both domains. Refolding of the same domains within the complete protein showed that domain–domain interactions direct the folding of both domains, but in an asymmetric way. Folding of the N domain was already altered within 1 msec, while detectable changes in the folding of the C domain occurred only 60–100 msec after initiating refolding. All mutants showed a hyperfluorescent kinetic intermediate. Both the disappearance of this intermediate and the completion of the folding were significantly faster in the individual N domain than in the complete protein. On the contrary, folding of the individual C domain was slower than in the complete protein. The presence of the C domain directs the refolding of the N domain along a completely different pathway than that of the individual N domain, while folding of the individual C domain follows the same path as within the complete protein.

Abstract: Interest in the structural and dynamic properties of unfolded proteins has increased in recent years owing to continued interest in protein folding and misfolding. Knowledge of the unfolded state under native conditions is particularly important for obtaining a complete picture of the protein folding process. The C-terminal domain of protein L9 is a globular alpha, beta protein with an unusual mixed parallel and antiparallel beta-strand structure. The folding kinetics and equilibrium unfolding of CTL9 strongly depend on pH, and follow a simple two state model. Both the native and the unfolded state can be significantly populated at pH 3.8 in the absence of denaturant, allowing the native state and the unfolded state to be characterized under identical conditions. Backbone (15)N, (13)C, (1)H and side-chain (13)C(beta), (1)H(beta) chemical shifts, amide proton NOEs, and (15)N R(2) relaxation rates were obtained for the two conformational states at pH 3.8. All the data indicate that the pH 3.8 native state is well folded and is similar to the native state at neutral pH. There is significant residual structure in the pH 3.8 unfolded state. The regions corresponding to the two native state alpha-helices show strong preference to populate helical phi and psi angles. The segment that connects alpha-helix 2 and beta-strand 2 has a significant tendency to form non-native alpha-helical structure. Comparison with the pH 2.0 unfolded state and the urea unfolded state indicates that the tendency to adopt both native and non-native helical structure is stronger at pH 3.8, demonstrating that the unfolded state of CTL9 under native-like conditions is more structured. The implications for the folding of CTL9 are discussed.

Abstract: Kinetic studies of the early events in cytochrome c folding are reviewed with a focus on the evidence for folding intermediates on the submillisecond timescale. Evidence from time-resolved absorption, circular dichroism, magnetic circular dichroism, fluorescence energy and electron transfer, small-angle X-ray scattering and amide hydrogen exchange studies on the t ≤ 1 ms timescale reveals a picture of cytochrome c folding that starts with the ~ 1-μs conformational diffusion dynamics of the unfolded chains. A fractional population of the unfolded chains collapses on the 1 – 100 μs timescale to a compact intermediate IC containing some native-like secondary structure. Although the existence and nature of IC as a discrete folding intermediate remains controversial, there is extensive high time-resolution kinetic evidence for the rapid formation of IC as a true intermediate, i.e., a metastable state separated from the unfolded state by a discrete free energy barrier. Final folding to the native state takes place on millisecond and longer timescales, depending on the presence of kinetic traps such as heme misligation and proline mis-isomerization. The high folding rates observed in equilibrium molten globule models suggest that IC may be a productive folding intermediate. Whether it is an obligatory step on the pathway to the high free energy barrier associated with millisecond timescale folding to the native state, however, remains to be determined.

Abstract: Most of fundamental studies on protein folding have been performed with small globular proteins consisting of a single domain. In vitro many of these proteins are well characterized by a reversible two-state folding scheme. However, the majority of proteins in the cell belong to the class of larger multi-domain proteins that often unfold irreversibly under in vitro conditions. This makes folding studies difficult or even impossible. In spite of these problems for many multi-domain proteins, folding has been investigated by classical refolding. Co-translational folding of nascent polypeptide chains when synthesized by ribosomes has also been studied. Single molecule techniques represent a promising approach for future studies on the folding of multi-domain proteins, and tremendous advances have been made in these techniques in recent years. In particular, fluorescence-based methods can contribute significantly to an understanding of the fundamental principles of multi-domain protein folding.

Abstract: The villin headpiece subdomain, HP36, is the smallest naturally occurring protein that folds cooperatively. Its small size, rapid folding, and simple three-helix topology have made it an extremely popular system for computational studies of protein folding. The role of unfolded state structure in rapid folding is an area of active investigation, but relatively little is known about the properties of unfolded states under native conditions. A peptide fragment, HP21, which contains the first and second helices of HP36 has been shown to be a good model for structure in the unfolded state of the intact domain but a detailed description of the conformational propensities of HP21 is lacking and the balance between native and nonnative interactions is not known. A series of three-dimensional NMR experiments were performed on 13C, 15N-labeled HP21 to investigate in detail its conformational propensities. Analysis of 13Cα, 13Cβ, 13CO chemical shifts, Δδ13Cα − Δδ13Cβ secondary shifts, the secondary structure propensity scores, NOEs, 15N R2 values and comparison of experimental chemical shifts with those of HP36 and with chemical shifts calculated using the SHIFTS and SHIFTX programs all indicate that there is significant native like structure in the HP21 ensemble, and thus by implication in the unfolded state of HP36.

Abstract: The effect of surface tethering on the folding mechanism of the src-SH3 protein domain was investigated using a coarse-grained Gō-type protein model. The protein was tethered at various locations along the protein chain and the thermodynamics and kinetics of folding were studied using replica exchange and constant temperature Langevin dynamics. Our simulations reveal that tethering in a structured part of the transition state can dramatically alter the folding mechanism, while tethering in an unstructured part leaves the folding mechanism unaltered as compared to bulk folding. Interestingly, there is only modest correlation between the tethering effect on the folding mechanism and its effect on thermodynamic stability and folding rates. We suggest locations on the protein at which tethering could be performed in single-molecule experiments so as to leave the folding mechanism unaltered from the bulk.

Abstract: To understand the role of sequence connectivity in protein folding pathways, we explored by Phi-value analysis the folding pathway of an engineered circularly permuted PDZ domain. This variant has the same sequence connectivity as naturally occurring circularly permuted PDZ domains and displays a symmetrical distribution of secondary structure elements (i.e., beta beta alpha beta beta alpha beta beta) while maintaining the same tertiary interactions of the well-characterized second PDZ domain from PTP-BL (PDZ2). Reliable Phi values were obtained for both a low-energy intermediate and the late rate-limiting transition state, allowing a description of both early and late events in folding. A comparison with Phi values obtained for wild-type PDZ2 reveals that while the structure of the late transition state is robust and unaffected by circular permutation, the folding intermediate is stabilized by a different nucleus involving residues located at the new N- and C-termini. The results suggest that folding is driven by competing nuclei whose stabilities may be selectively tuned by circular permutation.

Abstract: The structure of proteins as well as their folding/unfolding equilibrium are commonly attributed to H-bonding and hydrophobic interactions. We have used the molecular dynamic simulations in an explicit water environment based on the standard empirical potential as well as more accurately (and thus also more reliably) on the QM/MM potential. The simulations where the dispersion term was suppressed have led to a substantial change of the tryptophan-cage protein structure (unfolded structure). This structure cannot fold without the dispersion energy term, whereas, if it is covered fully, the system finds its native structure relatively quickly. This implies that after such physical factors as temperature and pH, the dispersion energy is an important factor in protein structure determination as well as in the protein folding/unfolding equilibrium. The loss of dispersion also affected the R-helical structure. On the other hand, weakening the electrostatic interactions (and thus H-bonding) affected the R-helical structure only to a minor extent.

Abstract: Successful ab initio folding of proteins with both α-helix and β-sheet requires a delicate balance among a variety of forces in the simulation model, which may explain that the successful folding of any α/β proteins to within experimental error has yet to be reported. Here we demonstrate that it is an achievable goal to fold α/β proteins with a force field emphasizing the balance between the two major secondary structures. Using our newly developed force field, we conducted extensive ab initio folding simulations on an α/β protein full sequence design (FSD) employing both conventional molecular dynamics and replica exchange molecular dynamics in combination with a generalized-Born solvation model. In these simulations, the folding of FSD to the native state with high population (>64.2%) and high fidelity (Cα-Root Mean Square Deviation of 1.29 A for the most sampled conformation when compared to the experimental structure) was achieved. The folding of FSD was found to follow two pathways. In the major pathway, the folding started from the formation of the helix. In the minor pathway, however, folding of the β-hairpin started first. Further examination revealed that the helix initiated from the C-terminus and propagated toward the N-terminus. The formation of the hydrophobic contacts coincided with the global folding. Therefore the hydrophobic force does not appear to be the driving force of the folding of this protein.

Abstract: Topology-based interaction potentials are simplified models that use the native contacts in the folded structure of a protein to define an energetically unfrustrated folding funnel. They have been widely used to analyze the folding transition and pathways of different proteins through computer simulations. Obviously, they need a reliable, experimentally determined folded structure to define the model interactions. In structures elucidated through NMR spectroscopy, a complex treatment of the raw experimental data usually provides a series of models, a set of different conformations compatible with the available experimental data. Here, we use an efficient coarse-grained simulation technique to independently consider the contact maps from every different NMR model in a protein whose structure has been resolved by the use of NMR spectroscopy. For lambda-Cro repressor, a homodimeric protein, we have analyzed its folding characteristics with a topology-based model. We have focused on the competition between the folding of the individual chains and their binding to form the final quaternary structure. From 20 different NMR models, we find a predominant three-state folding behavior, in agreement with experimental data on the folding pathway for this protein. Individual NMR models, however, show distinct characteristics, which are analyzed both at the level of the interplay between tertiary/quaternary structure formation and also regarding the thermal stability of the tertiary structure of every individual chain.

Abstract: An in vitro experiment with protein folding is typically initiated with 6 M GdmCl-denatured proteins, which are generally considered fully unfolded. However, studies conducted by various laboratories have shown that many 6 M GdmCl-denatured proteins are structurally heterogeneous and still retain nativelike residual structures. The extent of conformational heterogeneity of the 6 M GdmCl-denatured protein has significant implications for the folding landscape as well as the interpretation of the observed early stage folding mechanism. Using the method of disulfide scrambling, we are able to gain rough insight into the diverse structural properties of 6 M GdmCl-denatured proteins. It demonstrates that most 6 M GdmCl-denatured proteins are approximately fully denatured, but partially unfolded. Most of them comprise diverse conformational isomers. We review here the cumulative evidence obtained from various laboratories and also provide experimental data obtained in our laboratory.

Abstract: An increasing number of protein structures are found to encompass multiple folding nuclei, allowing their structures to be formed by several competing pathways. A typical example is the ribosomal protein S6, which comprises two folding nuclei (σ1 and σ2) defining two competing pathways in the folding energy landscape: σ1 → σ2 and σ2 → σ1. The balance between the two pathways, and thus the order of folding events, is easily controlled by circular permutation. In this study, we make use of this ability to manipulate the folding pathway to demonstrate that the dynamic motions of the S6 structure are independent of how the protein folds. The HD-exchange protection factors remain the same upon complete reversal of the folding order. The phenomenon arises because the HD-exchange motions and the high-energy excitations controlling the folding pathway occur at separated free-energy levels: the Boltzmann distribution of unproductive unfolding attempts samples all unfolding channels in parallel, even those that end up in excessively high barriers. Accordingly, the findings provide a simple rationale for how to interpret native-state dynamics without the need to invoke fluctuations off the normal unfolding reaction coordinate.

Abstract: Two different classes of experimental techniques exist by which protein folding mechanisms are ascertained. The first class, of which circular dichroism is an example, probes thermally-induced folding. The second class, which includes atomic force microscopy and optical tweezers, measures mechanically-induced folding. In this article, we investigate if proteins fold/unfold via the same mechanisms both thermally and mechanically. We do so using Ribonuclease H, a protein that has been shown to fold through a three-state mechanism using both types of experimental techniques. A detailed, molecular-level description of the states involved in thermal and mechanical folding shows that mechanisms for both types are globally similar, but small difference exist in the most unfolded conformations. Comparison to previous work suggests a universal folding behavior for proteins with a core helical bundle.

Abstract: This chapter describes the approaches and considerations necessary for extension of current protein folding methods to the equilibrium and kinetic reactions of oligomeric proteins, using dimers as the primary example. Spectroscopic and transport methods to monitor folding and unfolding transitions are summarized. The data collection and analyses to determine protein stability and kinetic folding mechanisms are discussed in the context of the additional dimension of complexity that arises in higher order folding processes, compared to first order monomeric proteins. As a case study to illustrate the data analysis process, equilibrium, and kinetic data are presented for SmtB, a homodimeric DNA-binding protein from Synechococcus PCC7942.