Abstract: Proteins, the workhorses of living systems, are constructed from chains of amino acids, which are synthesized in the cell based on the instructions of the genetic code and then folded into working proteins. The time for folding varies from microseconds to hours. What controls the folding rate is hotly debated. We postulate here that folding has the same temperature dependence as the α-fluctuations in the bulk solvent but is much slower. We call this behavior slaving. Slaving has been observed in folded proteins: Large-scale protein motions follow the solvent fluctuations with rate coefficient kα but can be slower by a large factor. Slowing occurs because large-scale motions proceed in many small steps, each determined by kα. If conformational motions of folded proteins are slaved, so a fortiori must be the motions during folding. The unfolded protein makes a Brownian walk in the conformational space to the folded structure, with each step controlled by kα. Because the number of conformational substates in the unfolded protein is extremely large, the folding rate coefficient, kf, is much smaller than kα. The slaving model implies that the activation enthalpy of folding is dominated by the solvent, whereas the number of steps nf = kα/kf is controlled by the number of accessible substates in the unfolded protein and the solvent. Proteins, however, undergo not only α- but also β-fluctuations. These additional fluctuations are local protein motions that are essentially independent of the bulk solvent fluctuations and may be relevant at late stages of folding.

Abstract: Minding your p's and q's has become as important to protein-folding theorists as it is for those being instructed in the rules of etiquette. To assess the quality of structural reaction coordinates in predicting the transition-state ensemble (TSE) of protein folding, we benchmarked the accuracy of four structural reaction coordinates against the kinetic measure Pfold, whose value of 0.50 defines the stochastic separatrix for a two-state folding mechanism. For two proteins that fold by a simple two-state mechanism, c-src SH3 and CI-2, the Φ-values of the TSEs predicted by native topology-based reaction coordinates (including Q, the fraction of native contacts) are almost identical to those of the TSE based on Pfold, with correlation coefficients of >0.90. For proteins with complex folding mechanisms that have especially broad, asymmetrical free energy barriers such as the designed 3-ankyrin repeating protein (3ANK) or proteins with distinct intermediates such as cyanovirin-N (CV-N), we show that the ensemble of structures with Pfold = 0.50 generally does not include the chemically relevant transition states. This weakness of Pfold limits its usefulness in protein folding studies. For such systems, elucidating the essential features of folding mechanisms requires using multiple reaction coordinates, although the number is still rather small. At the same time, for simple folding mechanisms, there is no indication of superiority for Pfold over structurally chosen and thermodynamically relevant reaction coordinates that correctly measure the degree of nativeness.

Abstract: The folding kinetics of a 16-residue beta-hairpin (trpzip4) and five mutants were studied by a laser-induced temperature-jump infrared method Our results indicate that mutations which affect the strength of the hydrophobic cluster lead to a decrease in the thermal stability of the beta-hairpin, as a result of increased unfolding rates For example, the W45Y mutant has a phi-value of approximately zero, implying a folding transition state in which the native contacts involving Trp45 are not yet formed On the other hand, mutations in the turn or loop region mostly affect the folding rate In particular, replacing Asp46 with Ala leads to a decrease in the folding rate by roughly 9 times Accordingly, the phi-value for D46A is determined to be approximately 077, suggesting that this residue plays a key role in stabilizing the folding transition state This is most likely due to the fact that the main chain and side chain of Asp46 form a characteristic hydrogen bond network with other residues in the turn region Taken together, these results support the folding mechanism we proposed before, which suggests that the turn formation is the rate-limiting step in beta-hairpin folding and, consequently, a stronger turn-promoting sequence increases the stability of a beta-hairpin primarily by increasing its folding rate, whereas a stronger hydrophobic cluster increases the stability of a beta-hairpin primarily by decreasing its unfolding rate In addition, we have examined the compactness of the thermally denatured and urea-denatured states of another 16-residue beta-hairpin, using the method of fluorescence resonance energy transfer Our results show that the thermally denatured state of this beta-hairpin is significantly more compact than the urea-denatured state, suggesting that the very first step in beta-hairpin folding, when initiated from an extended conformation, probably corresponds to a process of hydrophobic collapse

Abstract: The effects of macromolecular crowding on protein stability and folding kinetics have been studied using the recently developed 15N spin relaxation dispersion technique. By applying this method to a redesigned apocytochrome b562, the kinetics and thermodynamics of the protein folding processes in both the presence and the absence of crowding agents have been characterized. The result indicates that, even under the mild crowded environments (in the presence of 85 mg/mL of PEG 20K), the folding rate of the protein can speed up significantly while the unfolded rate remains unchanged within experimental error.

Abstract: For many decades, protein folding experimentalists have worked with no information about the time scales of relevant protein folding motions and without methods for estimating the height of folding barriers. Protein folding experiments have been interpreted using chemical models in which the folding process is characterized as a series of equilibria between two or more distinct states that interconvert with activated kinetics. Accordingly, the information to be extracted from experiments was circumscribed to apparent equilibrium constants and relative folding rates. Recent developments are changing this situation dramatically. The combination of fast-folding experiments with the development of analytical methods more closely connected to physical theory reveals that folding barriers in native conditions range from minimally high (approximately 14RT for the very slow folder AcP) to nonexistent. While slow-folding (i.e., > or = 1 ms) single-domain proteins are expected to fold in a two-state fashion, microsecond-folding proteins should exhibit complex behavior arising from crossing marginal or negligible folding barriers. This realization opens a realm of exciting opportunities for experimentalists. The free energy surface of a protein with a marginal (or no) barrier can be mapped using equilibrium experiments, which could resolve energetic factors from structural factors in folding. Kinetic experiments on these proteins provide the unique opportunity to measure folding dynamics directly. Furthermore, the complex distributions of time-dependent folding behaviors expected for these proteins might be accessible to single-molecule measurements. Here, we discuss some of these recent developments in protein folding, emphasizing aspects that can serve as a guide for experimentalists interested in exploiting this new avenue of research.

Abstract: Approximately 75% of eukaryotic proteins contain more than one so-called independently folding domain. However, there have been relatively few systematic studies to investigate the effect of interdomain interactions on protein stability and fewer still on folding kinetics. We present the folding of pairs of three-helix bundle spectrin domains as a paradigm to indicate how complex such an analysis can be. Equilibrium studies show an increase in denaturant concentration required to unfold the domains with only a single unfolding transition; however, in some cases, this is not accompanied by the increase in m value, which would be expected if the protein is a truly cooperative, all-or-none system. We analyze the complex kinetics of spectrin domain pairs, both wild-type and carefully selected mutants. By comparing these pairs, we are able to demonstrate that equilibrium data alone are insufficient to describe the folding of multidomain proteins and to quantify the effects that one domain can have on its neighbor.

Abstract: About 30% of proteins require cofactors for their proper folding. The effects of cofactors on the folding reaction have been investigated with α-lactalbumin as a model protein and metal ions as cofactors. Metal ions accelerate the refolding of α-lactalbumin by lessening the energy barrier between the molten globule state and the transition state, mainly by decreasing the difference of entropy between the two states. These effects are linked to metal ion binding to the protein in the native state. Hence, relationships between the metal affinities for the intermediate states and those for the native state are observed. Some residual specificity for the calcium ion is still observed in the molten globule state, this specificity getting closer in the transition state to that of the native state. The comparison between kinetic and steady-state data in association with the Φ value method indicates the binding of the metal ions on the unfolded state of α-lactalbumin. Altogether, these results provide insight into cofactor effects on protein folding. They also suggest new possibilities to investigate the presence of residual native structures in the unfolded state of protein and the effects of such structures on the protein folding reaction and on protein stability.

Abstract: The F61A/A90G mutant of a redesigned form of apocytochrome b562 folds by an apparent two-state mechanism We have used the pressure dependence of 15N NMR relaxation dispersion rate profiles to study the changes in volumetric parameters that accompany the folding reaction of this protein at 45 °C The experiments were performed under conditions where the folding/unfolding equilibrium could be studied at each pressure without addition of denaturants The exquisite sensitivity of the methodology to small changes in folding/unfolding rates facilitated the use of relatively low-pressure values (between 1 and 270 bar) so that pressure-induced changes to the unfolded state ensemble could be minimized A volume change for unfolding of −81 mL/mol is measured (at 1 bar), a factor of 14 larger (in absolute value) than the volume difference between the transition state ensemble (TSE) and the unfolded state Notably, the changes in the free energy difference between folded and unfolded states and in the activation fr

Abstract: Src homology 3 (SH3) domains are small modules that are thought to fold via a two-state mechanism, without the accumulation of significant populations of intermediate states. Relaxation dispersion NMR studies of the folding of G48V and G48M mutants of the Fyn SH3 domain have established that, at least for these modules, folding proceeds through the formation of a transient on-pathway intermediate with an equilibrium population of 1-2% that can be readily detected [Korzhnev, D. M., et al. (2004) Nature 430, 586-590]. To investigate the generality of this result, we present an (15)N relaxation dispersion NMR study of a pair of additional SH3 domains, including a G48V mutant of a stabilized Abp1p SH3 domain that shares 36% sequence identity with the Fyn SH3 module, and a A39V/N53P/V55L mutant Fyn SH3 domain. A transient folding intermediate is detected for both of the proteins studied here, and the dispersion data are well fit to a folding model of the form F I U, where F, I, and U correspond to folded, intermediate, and unfolded states, respectively. The temperature dependencies of the folding/unfolding rate constants were obtained so that the thermodynamic properties of each of F, I, and U could be established. The detection of I states in folding pathways of all SH3 domains examined to date via relaxation dispersion NMR spectroscopy indicates that such intermediates may well be a conserved feature in the folding of such domains in general but that their transient nature along with their low population makes detection difficult using more well-established approaches to the study of folding.

Abstract: Time-correlated single photon counting (TCSPC) was combined with fluorescence correlation spectroscopy (FCS) to study the transition between acid-denatured states and the native structure of cytochrome c (Cyt c) from Saccharomyces cerevisiae. The use of these techniques in concert proved to be more powerful than either alone, yielding a two-dimensional picture of the folding energy landscape of Cyt c. TCSPC measured the distribution of distances between the heme of the protein and a covalently attached dye molecule at residue C102 (one folding reaction coordinate), whereas FCS measured the hydrodynamic radius (a second folding reaction coordinate) of the protein over a range of pH values. These two independent measurements provide complimentary information regarding protein conformation. We see evidence for a well defined folding intermediate in the acid renaturation folding pathway of this protein reflected in the distribution of lifetimes needed to fit the TCSPC data. Moreover, FCS studies revealed this intermediate state to be in dynamic equilibrium with unfolded structures, with conformational fluctuations into and out of this intermediate state occurring on an ≈30-μs time scale.

Abstract: Search and study of the general principles that govern kinetics and thermodynamics of protein folding generate a new insight into the factors controlling this process. Here, based on the known experimental data and using theoretical modeling of protein folding, we demonstrate that there exists an optimal relationship between the average conformational entropy and the average energy of contacts per residue-that is, an entropy capacity-for fast protein folding. Statistical analysis of conformational entropy and number of contacts per residue for 5829 protein structures from four general structural classes (all-alpha, all-beta, alpha/beta, alpha+beta) demonstrates that each class of proteins has its own class-specific average number of contacts (class alpha/beta has the largest number of contacts) and average conformational entropy per residue (class all-alpha has the largest number of rotatable angles phi, psi, and chi per residue). These class-specific features determine the folding rates: alpha proteins are the fastest folding proteins, then follow beta and alpha+beta proteins, and finally alpha/beta proteins are the slowest ones. Our result is in agreement with the experimental folding rates for 60 proteins. This suggests that structural and sequence properties are important determinants of protein folding rates.

Abstract: The volumetric properties associated with protein folding transitions reflect changes in protein packing and hydration of the states that participate in the folding reaction. Here, NMR spin relaxation techniques are employed to probe the folding−unfolding kinetics of two SH3 domains as a function of pressure so that the changes in partial molar volumes along the folding pathway can be measured. The two domains fold with rates that differ by ∼3 orders of magnitude, so their folding dynamics must be probed using different NMR relaxation experiments. In the case of the drkN SH3 domain that folds via a two-state mechanism on a time scale of seconds, nitrogen magnetization exchange spectroscopy is employed, while for the G48M mutant of the Fyn SH3 domain where the folding occurs on the millisecond time scale (three-step reaction), relaxation dispersion experiments are utilized. The NMR methodology is extremely sensitive to even small changes in equilibrium and rate constants, so reliable estimates of partial m...

Abstract: Preface Molecular Basis, Biological and Clinical Effects of Protein Misfolding and Aggregation Folding Intermediates: Turning Points to the Native State, Amyloid Fibrils and Aggregation Mechanism of the Chaperone-Like Activity Existence of Condensed Structures in Denatured Lysozyme and their Contributions to the Folding Secondary Structure of Proteins: Implications for Folding, Function and Protein Structure Prediction Electronic Aspects of Protein Folding: Substituent Effects of Amino Acids in Dipeptides Salient Features of Residue Contacts in Protein Structures for Determining their Folding and Stability Index.

Abstract: Collagen, the most plentiful protein in mammals, constitutes approximately one quarter of all proteins in the human body (Stryer 1988). A variety of tissues, such as blood vessels, bone, and cartilage, owe their stability and tensile strength to collagen. The unique properties of collagen are related to its distinctive structure and amino acid composition. Unlike the compact, roughly spherical structures of globular proteins, collagen monomers are composed of three polypeptide chains that fold into a triple-helical rod (Brodsky and Persikov 2005). All side chains within this structure are solvent exposed—a stark contrast to globular structures, which have a significant number of their side chains buried within a hydrophobic core. This unique conformation imposes significant constraints on the sequence of each polypeptide chain. Given the close packing of amino acids near the common axis of the triple-helical rod, every third position in the amino acid sequence must be a Gly residue. Consequently, mature collagen sequences consist of triplets of the form X1-X2-Gly, where X1 and X2 are frequently Pro and Hyp, respectively (Stryer 1988). An additional defining characteristic of the structure of collagen is the hydrogen bonding pattern of its main chain. The backbone carbonyl of residue X1 in each triplet directly hydrogen bonds to a second polypeptide chain, and the N-H group of the Gly in each triplet hydrogen bonds to the other polypeptide chain (Brodsky and Persikov 2005). NMR and X-ray crystallographic studies on collagen-like model peptides have clarified many aspects of the mechanism of collagen folding and have been instrumental in deciphering how sequence variation affects the structure of collagen (Buevich and Baum 2001). Kinetic measurements of folding rates for collagen-like peptides suggest that folding consists of several steps, which include the formation of a triple-helical region near the C terminus—a C-terminal nucleated trimer—followed by propagation of the triple-helical structure from the C terminus to the N terminus in a “zipper-like” mechanism (Buevich et al. 2000; Buevich and Baum 2001; Bachmann et al. 2005). Bottlenecks in the folding pathway arise from two factors: (1) the formation of a C-terminal nucleated trimer, and (2) cis-trans isomerization of Gly-Pro bonds (Buevich and Baum 2001; Bachmann et al. 2005). Mutations that occur at positions normally occupied by Gly residues can alter this folding pathway and lead to the formation of structures that differ from the classic triple-helical fold. For instance, peptides containing a Gly → Ala mutation form triple-helical structures that contain a local bulge at the site of the mutation and a disruption of the normal collagen hydrogen bond pattern (Bella et al. 1994). Mutants containing amino acids with larger side chains can lead to more significant alterations in the structure (Beck et al. 2000; Buevich and Baum 2001). As a number of mutations have been linked to important connective tissue diseases, a thorough understanding of factors that affect the folding of collagen is of particular interest (Prockop and Ala-Kokko 2004). Osteogenesis imperfecta (OI) is one such connective-tissue disorder associated with missense mutations that result in substitutions for Gly residues (Rauch and Glorieux 2004). The clinical manifestations of OI vary greatly depending on the type of mutation and where it occurs in the sequence of type I collagen (Prockop and Ala-Kokko 2004). Patients with severe forms of OI suffer a myriad of symptoms, which may include multiple fractures, hearing loss, and heart failure secondary to valvular heart disease (Prockop and Ala-Kokko 2004). The most severe forms of OI are lethal in the perinatal period (Prockop and Ala-Kokko 2004). A number of studies have identified patients with both nonlethal and lethal forms of OI who have missense mutations resulting in Gly → Ser substitutions in type I collagen (Westerhausen et al. 1990; Bateman et al. 1992; Prockop and Ala-Kokko 2004). To better understand the folding mechanism of native collagen and of mutants associated with OI at an atomistic level of detail, we calculated folding trajectories of a peptide model of native collagen, (Pro-Hyp-Gly)10, which we henceforth refer to as peptide 1, and of a mutant bearing a Gly → Ser substitution that models a mutation found in α1(I) chains of some patients with OI, (Pro-Hyp-Gly)3-Pro-Hyp-Ser-(Pro-Hyp-Gly)6. We refer to this latter sequence as peptide 2. Moreover, to fully explore the role that solvent has in the folding pathway, folding simulations were performed with explicit solvent. The formation of triple-helical structures from individual peptide chains occurs on a time scale that is not readily accessible from the standpoint of molecular simulations (Buevich and Baum 2001). Therefore, to simplify the calculations, we focus on the latter stages of folding using an all-trans model of peptides 1 and 2 that contains a C-terminal nucleated trimer. While this greatly reduces the computational complexity of the problem, folding to a triple-helical structure from this unfolded state likely occurs on a time scale that cannot be easily achieved using conventional molecular dynamics (MD) simulations with explicit solvent (Bachmann et al. 2005). Consequently, to promote folding within a shorter period of time, we employ a technique which enables us to observe a folding event during a MD trajectory. In the biased MD (BMD) approach, a gentle bias is introduced during MD simulations to favor the formation of a prespecified conformation, that is, the target (Harvey and Gabb 1993; Marchi and Ballone 1999; Paci and Karplus 1999). This is accomplished by (1) selecting fluctuations during a molecular simulation that take the system closer to the target conformation, and (2) by associating a small penalty to fluctuations that take the system away from the target. In the current study, we bias the trajectory, which begins with the unfolded structure, toward folded conformations. The method is attractive because the gentle bias does not significantly affect the short-term dynamics of the system and, by design, the bias is not introduced when the system undergoes fluctuations that naturally cause the protein to fold (Paci and Karplus 1999). In addition, the use of a gentle biasing function in folding simulations with explicit solvent is desirable as this helps to ensure that the solvent molecules will relax about a changing protein structure. The approach has been implemented in CHARMM and has been applied to a number of different systems (Paci and Karplus 1999; Paci et al. 2001, 2005; Morra et al. 2003). Using a combination of BMD and standard umbrella sampling, we develop a detailed picture of collagen folding that yields additional insights into the folding mechanism of collagen.

Abstract: The focal adhesion target (FAT) domain of focal adhesion kinase has a four-helix bundle structure. Based on a hydrogen exchange-constrained computer simulation study and some indirect experimental results, it has been suggested that a partially unfolded state of the FAT domain with the N-terminal helix unfolded plays an important role in its biological function. Here, using a native-state hydrogen exchange method, we directly detected an intermediate with the N-terminal helix unfolded in a mutant (Y925E) of the FAT domain. In addition, kinetic folding studies on the FAT domain suggest that this intermediate exists on the native side of the rate-limiting transition state for folding. These results provide more direct evidence of the existence of the proposed intermediate and help to understand the folding mechanism of small single domain proteins.

Abstract: Molecular dynamics simulations can be used to reveal the detailed conformational behaviors of peptides and proteins. By comparing fragment and full-length protein simulations, we can investigate th...