Abstract: We report single-molecule folding studies of a small, single-domain protein, chymotrypsin inhibitor 2 (CI2). CI2 is an excellent model system for protein folding studies and has been extensively studied, both experimentally (at the ensemble level) and theoretically. Conformationally assisted ligation methodology was used to synthesize the proteins and site-specifically label them with donor and acceptor dyes. Folded and denatured subpopulations were observed by fluorescence resonance energy transfer (FRET) measurements on freely diffusing single protein molecules. Properties of these subpopulations were directly monitored as a function of guanidinium chloride concentration. It is shown that new information about different aspects of the protein folding reaction can be extracted from such subpopulation properties. Shifts in the mean transfer efficiencies are discussed, FRET efficiency distributions are translated into potentials, and denaturation curves are directly plotted from the areas of the FRET peaks. Changes in stability caused by mutation also are measured by comparing pseudo wild-type CI2 with a destabilized mutant (K17G). Current limitations and future possibilities and prospects for single-pair FRET protein folding investigations are discussed.

Abstract: I attempt to reconcile apparently conflicting factors and mechanisms that have been proposed to determine the rate constant for two-state folding of small proteins, on the basis of general features of the structures of transition states. Φ-Value analysis implies a transition state for folding that resembles an expanded and distorted native structure, which is built around an extended nucleus. The nucleus is composed predominantly of elements of partly or well-formed native secondary structure that are stabilized by local and long-range tertiary interactions. These long-range interactions give rise to connecting loops, frequently containing the native loops that are poorly structured. I derive an equation that relates differences in the contact order of a protein to changes in the length of linking loops, which, in turn, is directly related to the unfavorable free energy of the loops in the transition state. Kinetic data on loop extension mutants of CI2 and α-spectrin SH3 domain fit the equation qualitatively. The rate of folding depends primarily on the interactions that directly stabilize the nucleus, especially those in native-like secondary structure and those resulting from the entropy loss from the connecting loops, which vary with contact order. This partitioning of energy accounts for the success of some algorithms that predict folding rates, because they use these principles either explicitly or implicitly. The extended nucleus model thus unifies the observations of rate depending on both stability and topology.

Abstract: A lattice model with side chains was used to investigate protein folding with computer simulations. In this model, we rigorously demonstrate the existence of a specific folding nucleus. This nucleus contains specific interactions not present in the native state that, when weakened, slow folding but do not change protein stability. Such a decoupling of folding kinetics from thermodynamics has been observed experimentally for real proteins. From our results, we conclude that specific non-native interactions in the transition state would give rise to straight phi-values that are negative or larger than unity. Furthermore, we demonstrate that residue Ile 34 in src SH3, which has been shown to be kinetically, but not thermodynamically, important, is universally conserved in proteins with the SH3 fold. This is a clear example of evolution optimizing the folding rate of a protein independent of its stability and function.

Abstract: Two models have been proposed to describe the folding pathways of proteins The framework model assumes the initial formation of the secondary structures whereas the hydrophobic collapse model supposes their formation after the collapse of backbone structures To differentiate between these models for real proteins, we have developed a novel CD spectrometer that enables us to observe the submillisecond time frame of protein folding and have characterized the timing of secondary structure formation in the folding process of cytochrome c (cyt c) We found that ∼20% of the native helical content was organized in the first phase of folding, which is completed within milliseconds Furthermore, we suggest the presence of a second intermediate, which has α-helical content resembling that of the molten globule state Our results indicate that many of the α-helices are organized after collapse in the folding mechanism of cyt c

Abstract: The high structural resolution of the main transition states for the formation of native structure for the six small proteins of which Φ-values for a large set of mutants have become available, barstar, barnase, chymotrypsin inhibitor 2, Arc repressor, the src SH3 domain, and a tetrameric p53 domain reveals that for the first 5 of these proteins: (1) Residues that belong to regular secondary structure have a significantly larger average fraction of native structural consolidation than residues in loops; (2) on the other hand, secondary and tertiary structures have built up to the same degree, or at least a high degree, but nonuniformly distributed over the molecule; (3) the most consolidated parts of each protein molecule in the transition state cluster together, and these clusters contain a significantly higher percentage of residues that belong to regular secondary structure than the rest of the molecule. These observations further reconcile the framework model with the nucleation-condensation mechanism for folding: The amazing speed of protein folding can be understood as caused by the catalytic effect of the formation of clusters of residues which have particularly high preferences for the early formation of regular secondary structure in the presence of significant amounts of tertiary structure interactions. Proteins 2000;41:288–298. © 2000 Wiley-Liss, Inc.

Abstract: One of the outstanding questions in protein folding concerns the degree of heterogeneity in the folding transition state ensemble: does a protein fold via a large multitude of diverse “pathways,” or are the elements of native structure assembled in a well defined order? Herein, we build on previous point mutagenesis studies of the src SH3 by directly investigating the association of structural elements and the loss of backbone conformational entropy during folding. Double-mutant analysis of polar residues in the distal β-hairpin and the diverging turn indicates that the hydrogen bond network between these elements is largely formed in the folding transition state. A 10-glycine insertion in the n-src loop (which connects the distal hairpin and the diverging turn) and a disulfide crosslink at the base of the distal β-hairpin exclusively affect the folding rate, showing that these structural elements are nearly as ordered in the folding transition state as in the native state. In contrast, crosslinking the base of the RT loop or the N and C termini dramatically slows down the unfolding rate, suggesting that dissociation of the termini and opening of the RT loop precede the rate-limiting step in unfolding. Taken together, these results suggest that essentially all conformations in the folding transition state ensemble have the central three-stranded β-sheet formed, indicating that, for the src homology 3 domain, there is a discrete order to structure assembly during folding.

Abstract: Recent theories of protein folding suggest that individual proteins within a large ensemble may follow different routes in conformation space from the unfolded state toward the native state and vice versa. Herein, we introduce a new type of kinetics experiment that shows how different unfolding pathways can be selected by varying the initial reaction conditions. The relaxation kinetics of the major cold shock protein of Escherichia coli (CspA) in response to a laser-induced temperature jump are exponential for small temperature jumps, indicative of folding through a two-state mechanism. However, for larger jumps, the kinetics become strongly nonexponential, implying the existence of multiple unfolding pathways. We provide evidence that both unfolding across an energy barrier and diffusive downhill unfolding can occur simultaneously in the same ensemble and provide the experimental requirements for these to be observed.

Abstract: To test the hypothesis that the folding pathways of evolutionarily related proteins with similar three-dimensional structures but widely different sequences should be similar, the folding pathway of apoleghemoglobin has been characterized using stopped-flow circular dichroism, heteronuclear NMR pulse labeling techniques and mass spectrometry. The pathway of folding was found to differ significantly from that of a protein of the same family, apomyoglobin, although both proteins appear to fold through helical burst phase intermediates. For leghemoglobin, the burst phase intermediate exhibits stable helical structure in the G and H helices, together with a small region in the center of the E helix. The A and B helices are not stabilized until later stages of the folding process. The structure of the burst phase folding intermediate thus differs from that of apomyoglobin, in which stable helical structure is formed in the A, B, G and H helix regions.

Abstract: We have exploited a procedure to identify when hydrogen bonds (H-bonds) form under two-state folding conditions using equilibrium and kinetic deuterium/hydrogen amide isotope effects. Deuteration decreases the stability of equine cytochrome c and the dimeric and crosslinked versions of the GCN4-p1 coiled coil by ~0.5 kcal mol-1. For all three systems, the decrease in equilibrium stability is reflected by a decrease in refolding rates and a near equivalent increase in unfolding rates. This apportionment indicates that ~50% of the native H-bonds are formed in the transition state of these helical proteins. In contrast, an α/β protein, mammalian ubiquitin, exhibits a small isotope effect only on unfolding rates, suggesting its folding pathway may be different. These four proteins recapitulate the general trend that ~50% of the surface buried in the native state is buried in the transition state, leading to the hypothesis that H-bond formation in the transition state is cooperative, with α-helical proteins forming a number of H-bonds proportional to the amount of surface buried in the transition state.

Abstract: We have performed 128 folding and 45 unfolding molecular dynamics runs of chymotryp- sin inhibitor 2 (CI2) with an implicit solvation model for a total simulation time of 0.4 microseconds. Folding requires that the three-dimensional struc- ture of the native state is known. It was simulated at 300 K by supplementing the force field with a har- monic restraint which acts on the root-mean-square deviation and allows to decrease the distance to the target conformation. High temperature and/or the harmonic restraint were used to induce unfolding. Of the 62 folding simulations started from random conformations, 31 reached the native structure, while the success rate was 83% for the 66 trajecto- ries which began from conformations unfolded by high-temperature dynamics. A funnel-like energy landscape is observed for unfolding at 475 K, while the unfolding runs at 300 K and 375 K as well as most of the folding trajectories have an almost flat energy landscape for conformations with less than about 50% of native contacts formed. The sequence of events, i.e., secondary and tertiary structure forma- tion, is similar in all folding and unfolding simula- tions, despite the diversity of the pathways. Previ- ous unfolding simulations of CI2 performed with different force fields showed a similar sequence of events. These results suggest that the topology of the native state plays an important role in the folding process. Proteins 2000;39:252-260.

Abstract: The amino acid sequence and the folding motif are essential in determining the protein folding mechanism. The interplay between energetic (sequence-dependent) and topological (motif dependent) frustrations is investigated for two model beta-barrel proteins of the same native fold but with different interaction Hamiltonians. The nature of the folding transition state ensemble for both models is probed. The extent of structure in the transition state is determined by performing point mutations and recording their effect on the stability of the transition state through their φ values. The transition state shows more structural heterogeneity for the more frustrated sequence, a reflection of the increased roughness of the funneled energy landscape which restricts the number of pathways to the native state. The validity of the φ-analysis approach was assessed to be critically dependent on the degree of frustration of the model. The interpretation of φ values as a measure of the structure of the transition state breaks down for sequences with higher levels of frustration (lower cooperativity) in which a Kramers’ description of the folding reaction is no longer appropriate.

Abstract: The refolding kinetics of 13 proteins have been studied in the presence of 2,2,2-trifluoroethanol (TFE). Low concentrations of TFE increased the folding rates of all the proteins, whereas higher concentrations have the opposite effect. The extent of deceleration of folding correlates closely with similar effects of guanidine hydrochloride and can be related to the burial of accessible surface area during folding. For those proteins folding in a two-state manner, the extent of acceleration of folding correlates closely with the number of local backbone hydrogen bonds in the native structure. For those proteins that fold in a multistate manner, however, the extent of acceleration is much smaller than that predicted from the data for two-state proteins. These results support the concept that for two-state proteins the search for native-like contacts is a key aspect of the folding reaction, whereas the rate-determining steps for folding of multistate proteins are associated with the reorganization of stable structure within a collapsed state or with the search for native-like interactions within less structured regions.

Abstract: GroEL is an allosteric protein that facilitates protein folding in an ATP-dependent manner. Herein, the relationship between cooperative ATP binding by GroEL and the kinetics of GroE-assisted folding of two substrates with different GroES dependence, mouse dihydrofolate reductase (mDHFR) and mitochondrial malate dehydrogenase, is examined by using cooperativity mutants of GroEL. Strong intra-ring positive cooperativity in ATP binding by GroEL decreases the rate of GroEL-assisted mDHFR folding owing to a slow rate of the ATP-induced transition from the protein-acceptor state to the protein-release state. Inter-ring negative cooperativity in ATP binding by GroEL is found to affect the kinetic partitioning of mDHFR, but not of mitochondrial malate dehydrogenase, between folding in solution and folding in the cavity underneath GroES. Our results show that protein folding by this “two-stroke motor” is coupled to cooperative ATP binding.

Abstract: The folding mechanisms of two proteins in the family of intracellular lipid binding proteins, ileal lipid binding protein (ILBP) and intestinal fatty acid binding protein (IFABP), were examined. The structures of these all-β-proteins are very similar, with 123 of the 127 amino acids of ILBP having backbone and Cβ conformations nearly identical to those of 123 of the 131 residues of IFABP. Despite this structural similarity, the sequences of these proteins have diverged, with 23% sequence identity and an additional 16% sequence similarity. The folding process was completely reversible, and no significant concentrations of intermediates were observed by circular dichroism or fluorescence at equilibrium for either protein. ILBP was less stable than IFABP with a midpoint of 2.9 M urea compared to 4.0 M urea for IFABP. Stopped-flow kinetic studies showed that both the folding and unfolding of these proteins were not monophasic, suggesting that either multiple paths or intermediate states were present during th...

Abstract: Publisher Summary This chapter describes the relationship between sequence and structure in α-helices, β-hairpins, and β- sheets. First, there is an analysis regarding the individual conformational preferences of 20 amino acids. These tendencies could explain the conformational preferences detected in the random coil state, as well as the secondary structure propensities of the 20 amino acids. This is followed by a description of the extent of the knowledge regarding α-helices. The α-helix is the best known and most easily recognized of the polypeptide regular structures. The chapter focuses on β-hairpins and β-sheets, about which much less is known. Finally, the importance of secondary structure elements in protein folding and stability is discussed. Kinetic analysis of proteins with stabilized α-helices demonstrates that an increase in the contribution of favorable local native interactions can, in some cases, optimize folding rates. Essentially, when a secondary structure element is folded in the transition state, its stabilization will accelerate folding. As in the case of the protein stabilization the increase in folding speed will not be equivalent to the stabilization of the secondary structure element, since the denatured state will also be stabilized.

Abstract: Publisher Summary This chapter presents both the general and specific results obtained from the studies of equilibrium folding intermediates and their relationship to the kinetic pathway of protein folding. A detailed comparison between various equilibrium ensembles and kinetic intermediates suggests that such equilibrium studies can yield important and insightful information about where in conformational space a protein chain can go and how it gets there. As with many properties of proteins, the relationship between the partially folded ensembles populated at equilibrium and those transiently populated during the folding reaction depends on the particular protein in question. Because they sample different regions of the energy landscape, kinetic and equilibrium intermediates will not necessarily be similar. For most proteins, however, there appears to be a dominant region that is the first to fold and/or the most stable. In other proteins, this trend is less clear, with only a subset of the interactions of equilibrium intermediates seen in kinetic intermediates. As more results and techniques become available, it helps to learn more about why a particular region of a protein may have a stronger influence on its folding and stability.

Abstract: We have measured changes in heat capacity, entropy, and enthalpy for each step in the folding reaction of CD2.d1 and evaluated the effects of core mutations on these properties. All wild-type and mutant forms fold through a rapidly formed intermediate state that precedes the rate-limiting transition state. Mutations have a pronounced effect on the enthalpy of both the intermediate and folded states, but in all cases a compensatory change in entropy results in a small net free-energy change. While the enthalpy change in the folded state can be attributed to a loss of van der Waals interactions, it has already been shown that changes in the stability of the intermediate are dominated by changes in secondary structure propensity [Lorch et al. (1999) Biochemistry 38, 1377-1385]. It follows that the thermodynamic basis of beta-propensity is enthalpic in origin. The effects of mutations on the enthalpy and entropy of the transition state are smaller than on the ground states. This relative insensitivity to mutation is discussed in the light of theories concerning the nature of the rate-limiting barrier in folding reactions.

Abstract: Traditionally, the empirical force field had great difficulties in simulating β-sheet folding. In the current study, we tested molecular dynamics simulations of β-sheet folding using a solvent-referenced potential. Three available β-sheet-forming synthetic peptides, TWIQNGSTKWYQNGSTKIYT, RGWSVQNGKYTNNGKTTEGR, and VFITSDPGKTYTEVDPGOKILQ, were simulated at their experimental temperatures. From extended initial conformations, all three peptides folded into β-sheet conformations. The calculated ratios of the β-structure from the 100 ns simulations were 26.5%, 17.8%, and 28.5%, respectively, for the three peptides. From different initial conformations, folding into β-sheets was also observed. With the same energy functions, the alanine-based peptide folded into helical conformations, demonstrating the sequence dependence of folding. During simulations, the β-sheet folding is usually initiated by the fast formation of turns. The three-strand compact structures with favorable inter-strand side-chain interactions...

Abstract: Abstract Understanding in detail the mechanism of protein folding in vitro requires all the conformational states encountered during folding to be known, including the rate limiting transition state. On initiating refolding, different proteins adopt a wide variety of different conformations very rapidly, before folding to the native state. Some protein:-, remain unfolded, others adopt partly folded conformations, while many adopt a compact but disordered state that has come to be known as the ‘molten globule’ (MG) (1). These relatively stable partly folded and MG conformations are frequently assumed to be crucial for directing the folding process, and much effort has gone into characterizing them (Chapter 6). This is usually difficult to achieve, since they are present only transiently. Determining the kinetic roles of such intermediate species, as to whether they are on-pathway intermediates, kinetically trapped, or simply the initially preferred conformational state of the unfolded protein under refolding conditions, is especially difficult with a complex process like protein folding. There is little direct evidence about the kinetic roles of most folding intermediates that have been detected (2, 3).

Abstract: Barnase is one of the few protein models that has been studied extensively for protein folding. Previous studies led to the conclusion that barnase folds through a very stable submillisecond intermediate (≈3 kcal/mol). The structure of this intermediate was characterized intensively by using a protein engineering approach. This intermediate has now been reexamined with three direct and independent methods. (i) Hydrogen exchange experiments show very small protection factors (≈2) for the putative intermediate, indicating a stability of ≈0.0 kcal/mol. (ii) Denaturant-dependent unfolding of the putative intermediate is noncooperative and indicates a stability less than 0.0 kcal/mol. (iii) The logarithm of the unfolding rate constant of native barnase vs. denaturant concentrations is not linear. Together with the measured rate (“I” to N), this nonlinear behavior accounts for almost all of the protein stability, leaving only about 0.3 kcal/mol that could be attributed to the rapidly formed intermediate. Other observations previously interpreted to support the presence of an intermediate are now known to have alternative explanations. These results cast doubts on the previous conclusions on the nature of the early folding state in barnase and therefore should have important implications in understanding the early folding events of barnase and other proteins in general.