Abstract: Proline cis−trans isomerization plays a key role in the rate-determining steps of protein folding. The energetic origin of this isomerization process is summarized, and the folding and unfolding of disulfide-intact bovine pancreatic ribonuclease A is used as an example to illustrate the kinetics and structural features of conformational changes from the heterogeneous unfolded state (consisting of cis and trans isomers of X-Pro peptide groups) to the native structure in which only one set of proline isomers is present.

Abstract: Publisher Summary Nuclear magnetic resonance (NMR) is unique in being able to provide detailed insights into the conformation of unfolded and partly folded proteins This chapter presents the structure and dynamics of unfolded states, that is, equilibrium NMR studies of apomyoglobin; characterization of proteins that are unstructured under nondenaturing conditions; and intrinsically unstructured proteins (coupled folding and binding events) NMR is the method of choice to determine the conformational preferences inherent in these domains NMR is also of the greatest utility in the elucidation of pathways of protein folding by allowing structural characterization of equilibrium and kinetic folding intermediates Through the application of state-of-the-art heteronuclear NMR methods, new insights have been obtained into the nature of the conformational ensemble and the dynamics of denatured proteins These studies show that the behavior of denatured proteins is typically far from that of a statistical random coil This new view of the denatured state provides a basis for understanding the earliest events that occur during protein folding

Abstract: During the evolution of proteins the pressure to optimize biological activity is moderated by a need for efficient folding. For most proteins, this is accomplished through spontaneous folding to a thermodynamically stable and active native state. However, in the extracellular bacterial alpha-lytic protease (alphaLP) these two processes have become decoupled. The native state of alphaLP is thermodynamically unstable, and when denatured, requires millennia (t1/2 approximately 1,800 years) to refold. Folding is made possible by an attached folding catalyst, the pro-region, which is degraded on completion of folding, leaving alphaLP trapped in its native state by a large kinetic unfolding barrier (t1/2 approximately 1.2 years). alphaLP faces two very different folding landscapes: one in the presence of the pro-region controlling folding, and one in its absence restricting unfolding. Here we demonstrate that this separation of folding and unfolding pathways has removed constraints placed on the folding of thermodynamically stable proteins, and allowed the evolution of a native state having markedly reduced dynamic fluctuations. This, in turn, has led to a significant extension of the functional lifetime of alphaLP by the optimal suppression of proteolytic sensitivity.

Abstract: The hydrogen exchange behavior of a four-helix bundle protein in low concentrations of denaturant reveals some partially unfolded forms that are significantly more stable than the fully unfolded state. Kinetic folding of the protein, however, is apparently two-state in the absence of the accumulation of early folding intermediates. The partially unfolded forms are either as folded as or more folded than the rate-limiting transition state and appear to represent the major intermediates in a folding and unfolding reaction. These results are consistent with the suggestion that partially unfolded intermediates may form after the rate-limiting step for small proteins with apparent two-state folding kinetics.

Abstract: Characterization of the unfolded state is essential for understanding the protein folding problem. In the unfolded state, a protein molecule samples vastly different conformations. Here I present a simple theoretical method for treating residual charge–charge interactions in the unfolded state. The method is based on modeling an unfolded protein as a Gaussian chain. After sampling over all conformations, the electrostatic interaction energy between two charged residues (separated by l peptide bonds) is given by W = 332(6/π)1/2[1 − π1/2xexp(x2)erfc(x)]/ɛd, where d = bl1/2 + s and x = κd/61/2. In unfolded barnase, the residual interactions lead to downward pKa shifts of ≈0.33 unit, in agreement with experiment. pKa shifts in the unfolded state significantly affect pH dependence of protein folding stability, and the predicted effects agree very well with experimental results on barnase and four other proteins. For T4 lysozyme, the charge reversal mutation K147E is found to stabilize the unfolded state even more than the folded state (1.39 vs. 0.46 kcal/mol), leading to the experimentally observed result that the mutation is net destabilizing for the folding. The Gaussian-chain model provides a quantitative characterization of the unfolded state and may prove valuable for elucidating the energetic contributions to the stability of thermophilic proteins and the energy landscape of protein folding.

Abstract: The thermodynamics and energetics of a 20-residue synthetic peptide with a stable three-stranded antiparallel beta-sheet fold are investigated by implicit solvent molecular dynamics (MD) at 330 K (slightly above the melting temperature in the model) and compared with previous simulation results at 360 K. At both temperature values, the peptide folds reversibly to the NMR solution conformation, irrespective of the starting conformation. The sampling of the conformational space (2.3 micros and 25 folding events at 330 K, and 3 micros and 50 folding events at 360 K) is sufficient to obtain a thermodynamic description of minima and transition states on the free energy surface, which is determined near equilibrium by counting populations. The free energy surface, plotted as a function of two-order parameters that monitor formation of either of the beta-hairpins, is similar at both temperature values. The statistically predominant folding pathway and its frequency (about two-thirds of the folding events) are the same at 330 K and 360 K. Furthermore, the main unfolding route is the reverse of the predominant folding pathway. The effective energy and its electrostatic and van der Waals contributions show a downhill profile at both temperatures, implying that the free energy barrier is of entropic origin and corresponds to the freezing of about two-thirds of the chain into a beta-hairpin conformation. The average folding rate is nearly the same at 330 K and 360 K, while the unfolding rate is about four times slower at 330 K than at 360 K. Taken together with previous MD analysis of alpha-helices and beta-hairpins, the present simulation results indicate that the free energy surface and folding mechanism of structured peptides have a weak temperature dependence.

Abstract: Tetraloops with the generic sequence GNRA are commonly found in RNA secondary structure, and interactions of such tetraloops with "receptors" elsewhere in RNA play important roles in RNA structure and folding. However, the contributions of tetraloop-receptor interactions specifically to the kinetics of RNA tertiary folding, rather than the thermodynamics of maintaining tertiary structure once folded, have not been reported. Here we investigate the role of the key GAAA tetraloop-receptor motif in folding of the P4-P6 domain of the Tetrahymena group I intron RNA. Insertions of one or more nucleotides into the tetraloop significantly disrupt the thermodynamics of tertiary folding; single-nucleotide insertions shift the folding free energy by 2-4 kcal/mol (¢¢G°'). The folding kinetics of several modified P4-P6 domains were determined by stopped-flow fluorescence spectroscopy, using an internally incorporated pyrene residue as the chromophore. In contrast to the thermodynamic results, the kinetics of Mg 2+ -induced folding were barely affected by the tetraloop modifications, with a ¢¢G q of 0.2-0.4 kcal/mol and a … value (ratio of the kinetic and thermodynamic contributions) of <0.1. These data indicate an early transition state for folding of P4-P6 with respect to forming the tetraloop-receptor contact, consistent with previous results for modifications elsewhere in P4-P6. We conclude that the GAAA tetraloop-receptor motif contributes little to the stabilization of the transition state for Mg 2+ -induced P4-P6 folding. Rather, the tetraloop- receptor motif acts to clamp the RNA once folding has occurred. This is the first report to correlate the kinetic and thermodynamic contributions of an important RNA tertiary motif, the GNRA tetraloop- receptor. The results are related to possible models for the Mg 2+ -induced folding of the P4-P6 RNA,

Abstract: The success of the protein folding process requires that the peptide chain find a structure that ensures the survival of its intramolecular H-bonds. In this work, we identify and model how water is hindered from invading and destroying the intramolecular H-bonds: a three-body protective association establishes itself when a hydrophobic residue approaches a pair of residues held by an amide–carbonyl H-bond. This proximity disrupts the water structure surrounding the backbone H-bond, driving water molecules away so they cannot solvate the backbone. These three-body contributions often compensate thermodynamically for concurrent two-body hydrophobic–polar mismatches. A previously-developed theoretical method to generate folding pathways is extended to reveal the role of three-residue correlations in stabilizing the collapse-inducing folding nucleus; successful computer runs exhibit their formation and how they protect and scaffold incipient secondary structure.

Abstract: Thirteen versions of a ‚-sheet protein have been constructed, each with a single, surface- exposed disulfide bridge. A comparison of folding kinetics, in oxidizing and reducing conditions, is used to elucidate the order in which ‚-strands become associated during the folding process and, hence, the relationship between topology and folding dynamics. In common with the wild-type molecule, all the proteins fold through a two-step (three state) mechanism with a rapidly formed intermediate which slowly converts to the native state. In a majority of cases, the bridge is seen to stabilize the folded state, and for five of the modified proteins, the additional stability is greater than 3 kcal/mol. Surprisingly, cross-links which connect ‚-strands which are distant in sequence predominantly stabilize the rapidly formed intermediate state, suggesting that these strand -strand interactions occur in the initial stages of folding. Cross-links which stabilize local hairpins have their major influence on the second, rate-determining step leading to significant enhancements in the folding rate. We find that enhancement of the folding rate in the second, rate-limiting step is correlated with a reduction in contact order in the same way as in naturally occurring proteins of different folds. The large increases in native-state stability resulting from the insertion of disulfide bridges on the surface of ‚-sheet structures have implications for enhancing the robustness of proteins by molecular engineering.

Abstract: Pectate lyase C (pelC) is a member of the class of proteins that possess a parallel beta-helix folding motif. A study of the kinetic folding mechanism is presented in this report. Kinetic circular dichroism (CD) and fluorescence have been used to observe changes in the structure of pelC as a function of time upon folding and unfolding. Three folding phases are observed with far-UV CD and four phases are observed with near-UV CD. The two slowest phases have relaxation times on the order of 21 and 46 s in aqueous buffer. Double-jump refolding assays and the measured activation enthalpies (16.0 and 21.2 kcal/mol for the respective slow phases) suggest that these two phases are the result of the slow cis-trans isomerization of prolyl-peptide bonds. We have determined that the earliest observed folding phase involves the formation of most, if not all, of the secondary structure with a relaxation time of 0.25 s. We also observed a phase by near-UV CD on the order of 0.25 s. This suggests that along with the appearance of secondary structure, some tertiary contacts are made. There is one kinetic phase observed in the near-UV CD and fluorescence that has no corresponding far-UV CD phase. This occurs with a relaxation time of 1.1 s. The temperature dependence of the natural log of the folding rate constant suggests that folding occurs via a sequential mechanism in which an on-pathway intermediate in rapid equilibrium with the unfolded protein is present. Semiempirical CD calculations support the idea that the beta-helix region of pelC forms in the fast kinetic phase, yielding near-native secondary and tertiary structures in that region. This is followed by the slower formation of the loop regions connecting individual strands of the beta-helix.

Abstract: An off-lattice all-atom (except nonpolar hydrogen) model of proteins based on Go j-type discontinuous interactions is developed and applied to study the folding thermodynamics of fragment B of Staphylococcal protein A. Unlike simpler CR based off-lattice models, which fold into a molten globule-like state from the coil state, the new model transits directly to the native state. The transition is strongly first-order-like and the dynamics of the native state is in approximate agreement with experimental data. The results suggest that a large well-packed solid core is the origin of the folding cooperativity of proteins.

Abstract: A hierarchic scheme of protein folding does not solve the Levinthal paradox since it cannot provide a simultaneous explanation for major features observed for protein folding: (i) folding within non-astronomical time, (ii) independence of the native structure on large variations in the folding rates of given protein under different conditions, and (iii) co-existence, in a visible quantity, of only the native and the unfolded molecules during folding of moderate size (single-domain) proteins. On the contrary, a nucleation mechanism can account for all these major features simultaneously and resolves the Levinthal paradox.

Abstract: In this paper we report that quasi-static-like processes, in which stable intermediates were introduced carefully and deliberately, may be used to reversibly unfold and refold purified native porcine growth hormone. Through circular dichroism (CD) and dynamic light scattering (DLS), we were able to study the secondary structure conformational changes, tertiary structure thermal stabilities, and the particle size distributions of both the intermediates and the final folded product. The CD data showed that the secondary structure was restored in the initial folding stage, whereas the tertiary structure within the protein was restored one step before the last folding stage, as elucidated by thermal stability experiments. DLS analysis suggested that the average hydrodynamic radii of the folding intermediates shrunk to nativelike size immediately after the first folding stage. Our data suggested that the denaturant-containing protein folding reaction is a first-order-like state transition process. This quasi-static-like process may be useful in the prevention of aggregate formation in protein purification and thus can be used in protein engineering to improve the overall yield from harvesting proteins.

Abstract: Publisher Summary This chapter discusses the role of protein disulfide isomerase (PDI) in the process of protein folding. PDI is the physiological catalyst in the formation of the native disulfide bond(s) of nascent polypeptides. In vitro PDI catalyzes oxidative formation, reduction, or isomerization of disulfide bonds depending on the redox potential of the environment. Folding of the peptide chain and formation of the native disulfide(s) of disulfide-containing proteins are two processes that are intimately connected and work in conjunction during folding. It is necessary for the target sequence to fold at least to some extent to bring the relevant sulfhydryl groups close enough in space for the formation of the correct disulfide bonds. The spontaneous folding and oxidation of sulfhydryl groups of the reduced and unfolded peptide chain are often slow processes in vitro. PDI promotes the folding of a peptide chain to a conformation favorable for native disulfide formation without the assistance of a chaperone prior to the formation of native disulfide bonds. Thus, in addition to the catalysis of the formation of native disulfides, PDI promotes the folding of a peptide to its native conformation as chaperones do.