Thermodynamical properties of reaction intermediates during apoplastocyanin folding in time domain

Two intermediates observed for the folding process of apoplastocyanin (apoPC) were investigated by using a photoinduced triggering system combined with the transient grating and transient lens methods. The thermodynamic quantities, enthalpy, heat capacity, partial volume, and thermal expansion volume changes during the protein folding reaction were measured in time domain for the first time. An interesting observation is the positive enthalpy changes during the folding process. This positive enthalpy change must be compensated by positive entropy changes, which could be originated from the dehydration effect of hydrophobic residues and/or the translational entropy gain of bulk water molecules. Observed negative heat capacity change was explained by the dehydration effect of hydrophilic residues and/or motional confinement of amino acid side chains and water molecules in apoPC. The signs of the volume change and thermal expansion volume were different for two processes and these changes were interpreted in terms of the different relative contributions of the hydration and the dehydration of the hydrophilic residues. These results indicated two-step hydrophobic collapses in the early stage of the apoPC folding, but the nature of the dynamics was different.     

Fast folding of a four-helical bundle protein

The FK506-FKBP12 binding-domain of the kinase FRAP (FRB) forms a classic up-down four-helical bundle. The folding pathway of this protein has been investigated using a combination of equilibrium and kinetic studies. The native state of the protein is stable with respect to the unfolded state by some 7 kcal mol(-1) at pH 6.0, 10 degrees C. A kinetic analysis of unfolding and refolding rate constants as a function of chemical denaturant concentration suggests that an intermediate state may be populated during folding at low concentrations of denaturant. The presence of this intermediate state is confirmed by refolding experiments performed in the presence of the hydrophobic dye 8-anilinonaphthalene-1 sulfonate (ANS). ANS binds to the partially folded intermediate state populated during the folding of FRB and undergoes a large change in fluorescence that can be detected using stopped-flow techniques. Analysis of the kinetic data suggests that the intermediate state is compact and it may even be a misfolded species that has to partially unfold before it can reach the transition state. Folding and unfolding rate constants in water are approximately 150-200 s(-1) and 0.005-0.06 s(-1), respectively, at neutral pH and 10 degrees C. The folding of FRB is somewhat slower than for other all-helical proteins, probably as a consequence of the formation of a metastable intermediate state. The folding rate constant in the absence of any populated intermediate can be estimated to be 8800 s(-1). Despite the presence of an intermediate state, which effectively slows folding, the protein still folds rapidly with a half-life of 5 ms at 10 degrees C. The dependence of the rate constants on denaturant concentration indicates that the transition state for folding is compact with some 80% of the surface area exposed in the unfolded state buried in the transition state. Data presented for FRB is compared with kinetic data obtained for other all-helical proteins.     

Oscillatory molecular driving force for protein folding at high concentration: a molecular simulation

This paper presents a Langevin dynamics simulation that suggests a novel way to fold protein at high concentration, a fundamental issue in neurodegenerative diseases in vivo and the production of recombinant proteins in vitro. The simulation indicates that the folding of a coarse-grained beta-barrel protein at high concentration follows the "collapse-rearrangement" mechanism but it yields products of various forms, including single proteins in the native, misfolded, and uncollapsed forms and protein aggregates. Misfolded and uncollapased proteins are the "nucleus" of the aggregates that also encapsulate some correctly folded proteins (native proteins). An optimum hydrophobic interaction strength (epsilon*(p)) between the hydrophobic beads of the model protein, which results from a compromise between the kinetics of collapse and rearrangement, is identified for use in increasing the rate of folding over aggregating. Increased protein concentration hinders the structural transitions in both collapse and rearrangement and thus favors aggregation. A new method for protein folding at high concentration is proposed, which uses an oscillatory molecular driving force (epsilon*(p)) to promote the dissociation of aggregates in the low epsilon*(p) regime while promoting folding at a high epsilon*(p). The advantage of this method in enhancing protein folding while depressing aggregation is illustrated by a comparison with the methods based on direct dilution or applying a denaturant gradient.     

A high-resolution probe of protein folding

Protein folding is a central problem in the biological sciences. To generate residue-specific information on the equilibrium folding of cytochrome c, we have semisynthesized the protein with specifically deuterated residues. The C-D bonds may be easily visualized in an otherwise transparent region of the IR spectra, even at high protein and denaturant concentrations. Plotted as a function of added guanidine hydrochloride denaturant, the absorption intensities reveal that the protein undergoes a conformational change at the protein-based ligand, Met80, which is then followed by a more global unfolding at 2.3 M denaturant. Deuteration and characterization of other residues in cytochrome c, or other protein of interest, should provide complete views of folding with residue specific detail that is capable of resolving even the most rapidly interconverting intermediates.     

Conformational changes during apoplastocyanin folding observed by photocleavable modification and transient grating

A new method to investigate the initial protein folding dynamics is developed based on a pulsed laser light triggering method and a unique transient grating method. The side chain of the cysteine residue of apoplastocyanin (apoPC) was site-specifically modified with a 4,5-dimethoxy-2-nitrobenzyl derivative, where the CD and 2D NMR spectra showed that the modified apoPC was unfolded. The substituent was cleaved with a rate of about 400 ns by photoirradiation, which was monitored by the disappearance of the absorption band at 355 nm and the increase in the transient grating signal. After a sufficient time from the photocleavage reaction, the CD and NMR spectra showed that the native beta-sheet structure was recovered. Protein folding dynamics was monitored in the time domain with the transient grating method from a viewpoint of the molecular volume change and the diffusion coefficient, both of which reflect the global structural change, including the protein-water interaction. The observed volume decrease of apoPC with a time scale of 270 micros is ascribed to the initial hydrophobic collapse. The increase in the diffusion coefficient (23 ms) is considered to indicate a change from an intermolecular to an intramolecular hydrogen bonding network. The initial folding process of apoPC is discussed based on these observations.     

NMR spectroscopic characterization of millisecond protein folding by transverse relaxation dispersion measurements

The cold shock protein CspB adopts its native and functional tertiary structure on the millisecond time scale. We employed transverse relaxation NMR methods, which allow a quantitative measurement of the cooperativity of this fast folding reaction on a residue basis. Thereby, chemical exchange contributions to the transverse relaxation rate (R(2)) were observed for every residue of CspB verifying the potential of this method to identify not only local dynamics but also to characterize global events. Toward this end, the homogeneity of the transition state of folding was probed by comparing Chevron plots (i.e., dependence of the apparent folding rate on the denaturant concentration) determined by stopped-flow fluorescence with Chevron plots of six residues acquired by R(2) dispersion experiments. The coinciding results obtained for probes at different locations in the three-dimensional structure of CspB indicate the ability and significance of transverse relaxation NMR to determine Chevron plots on a residue-by-residue basis providing detailed insights on the nature of the transition state of folding.     

Probing the transition state ensemble of a protein folding reaction by pressure-dependent NMR relaxation dispersion

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 degrees 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 1.4 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 free energy for folding were not linear with pressure. Thus, the difference in the isothermal compressibility upon unfolding (-0.11 mL mol(-1) bar(-1)) and, for the first time, the compressibility of the TSE relative to the unfolded state (0.15 mL mol(-1) bar(-1)) could be calculated. The results argue for a TSE that is collapsed but loosely packed relative to the folded state and significantly hydrated, suggesting that the release of water occurs after the rate-limiting step in protein folding. The notion of a collapsed and hydrated TSE is consistent with expectations based on earlier temperature-dependent folding studies, showing that the barrier to folding at 45 degrees C is entropic (Choy, W. Y.; Zhou, Z.; Bai, Y.; Kay, L. E. J. Am. Chem. Soc. 2005, 127, 5066-5072).     

Estimation of protein folding rate from Monte Carlo simulations and entropy capacity

The problem of protein self-organization is one of the most important problems of molecular biology nowadays. Despite the recent success in the understanding of general principles of protein folding, details of this process are yet to be elucidated. Moreover, the prediction of protein folding rates has its own practical value due to the fact that aggregation directly depends on the rate of protein folding. The time of folding has been calculated for 67 proteins with known experimental data at the point of thermodynamic equilibrium between unfolded and native states using a Monte Carlo model where each residue is considered to be either folded as in the native state or completely disordered. The times of folding for 67 proteins which reach the native state within the limit of 10(8) Monte Carlo steps are in a good correlation with the experimentally measured folding rate at the mid-transition point (the correlation coefficient is -0.82). Theoretical consideration of a capillarity model for the process of protein folding demonstrates that the difference in the folding rate for proteins sharing more spherical and less spherical folds is the result of differences in the conformational entropy due to a larger surface of the boundary between folded and unfolded phases in the transition state for proteins with more spherical fold. The capillarity model allows us to predict the folding rate at the same level of correlation as by Monte Carlo simulations. The calculated model entropy capacity (conformational entropy per residue divided by the average contact energy per residue) for 67 proteins correlates by about 78% with the experimentally measured folding rate at the mid-transition point.     

Theoretical analysis on changes in thermodynamic quantities upon protein folding: essential role of hydration

The free energy change associated with the coil-to-native structural transition of protein G in aqueous solution is calculated by using the molecular theory of solvation, also known as the three-dimensional reference interaction site model theory, to uncover the molecular mechanism of protein folding. The free energy is decomposed into the protein intramolecular energy, the hydration energy, and the hydration entropy. The folding is accompanied with a large gain in the protein intramolecular energy. However, it is almost canceled by the correspondingly large loss in the hydration energy due to the dehydration, resulting in the total energy gain about an order of magnitude smaller than might occur in vacuum. The hydration entropy gain is found to be a substantial driving force in protein folding. It is comparable with or even larger than the total energy gain. The total energy gain coupled with the hydration entropy gain is capable of suppressing the conformational entropy loss in the folding. Based on careful analysis of the theoretical results, the authors present a challenging physical picture of protein folding where the overall folding process is driven by the water entropy effect.     

New scenarios of protein folding can occur on the ribosome

Identifying and understanding the differences between protein folding in bulk solution and in the cell is a crucial challenge facing biology. Using Langevin dynamics, we have simulated intact ribosomes containing five different nascent chains arrested at different stages of their synthesis such that each nascent chain can fold and unfold at or near the exit tunnel vestibule. We find that the native state is destabilized close to the ribosome surface due to an increase in unfolded state entropy and a decrease in native state entropy; the former arises because the unfolded ensemble tends to behave as an expanded random coil near the ribosome and a semicompact globule in bulk solution. In addition, the unfolded ensemble of the nascent chain adopts a highly anisotropic shape near the ribosome surface and the cooperativity of the folding-unfolding transition is decreased due to the appearance of partially folded structures that are not populated in bulk solution. The results show, in light of these effects, that with increasing nascent chain length folding rates increase in a linear manner and unfolding rates decrease, with larger and topologically more complex folds being the most highly perturbed by the ribosome. Analysis of folding trajectories, initiated by temperature quench, reveals the transition state ensemble is driven toward compaction and greater native-like structure by interactions with the ribosome surface and exit vestibule. Furthermore, the diversity of folding pathways decreases and the probability increases of initiating folding via the N-terminus on the ribosome. We show that all of these findings are equally applicable to the situation in which protein folding occurs during continuous (non-arrested) translation provided that the time scales of folding and unfolding are much faster than the time scale of monomer addition to the growing nascent chain, which results in a quasi-equilibrium process. These substantial ribosome-induced perturbations to almost all aspects of protein folding indicate that folding scenarios that are distinct from those of bulk solution can occur on the ribosome.     

Simulation of chaperonin effect on protein folding: a shift from nucleation-condensation to framework mechanism

The iterative annealing mechanism (IAM) of chaperonin-assisted protein folding is explored in a framework of a well-established coarse-grained protein modeling tool, which enables the study of protein dynamics in a time-scale well beyond classical all-atom molecular mechanics. The chaperonin mechanism of action is simulated for two paradigm systems of protein folding, B domain of protein A (BdpA) and B1 domain of protein G (GB1), and compared to chaperonin-free simulations presented here for BdpA and recently published for GB1. The prediction of the BdpA transition state ensemble (TSE) is in perfect agreement with experimental findings. It is shown that periodic distortion of the polypeptide chains by hydrophobic chaperonin interactions can promote rapid folding and leads to a decrease in folding temperature. It is also demonstrated how chaperonin action prevents kinetically trapped conformations and modulates the observed folding mechanisms from nucleation-condensation to a more framework-like.     

Structural and thermodynamic investigations on the aggregation and folding of acylphosphatase by molecular dynamics simulations and solvation free energy analysis

Protein engineering method to study the mutation effects on muscle acylphosphatase (AcP) has been actively applied to describe kinetics and thermodynamics associated with AcP aggregation as well as folding processes. Despite the extensive mutation experiments, the molecular origin and the structural motifs for aggregation and folding kinetics as well as thermodynamics of AcP have not been rationalized at the atomic resolution. To this end, we have investigated the mutation effects on the structures and thermodynamics for the aggregation and folding of AcP by using the combination of fully atomistic, explicit-water molecular dynamics simulations, and three-dimensional reference interaction site model theory. The results indicate that the A30G mutant with the fastest experimental aggregation rate displays considerably decreased α1-helical contents as well as disrupted hydrophobic core compared to the wild-type AcP. Increased solvation free energy as well as hydrophobicity upon A30G mutation is achieved due to the dehydration of hydrophilic side chains in the disrupted α1-helix region of A30G. In contrast, the Y91Q mutant with the slowest aggregation rate shows a non-native H-bonding network spanning the mutation site to hydrophobic core and α1-helix region, which rigidifies the native state protein conformation with the enhanced α1-helicity. Furthermore, Y91Q exhibits decreased solvation free energy and hydrophobicity compared to wild type due to more exposed and solvated hydrophilic side chains in the α1-region. On the other hand, the experimentally observed slower folding rates in both mutants are accompanied by decreased helicity in α2-helix upon mutation. We here provide the atomic-level structures and thermodynamic quantities of AcP mutants and rationalize the structural origin for the changes that occur upon introduction of those mutations along the AcP aggregation and folding processes.     

Folding transition-state and denatured-state ensembles of FSD-1 from folding and unfolding simulations

Characterization of the folding transition-state ensemble and the denatured-state ensemble is an important step toward a full elucidation of protein folding mechanisms. We report herein an investigation of the free-energy landscape of FSD-1 protein by a total of four sets of folding and unfolding molecular dynamics simulations with explicit solvent. The transition-state ensemble was initially identified from unfolding simulations at 500 K and was verified by simulations at 300 K starting from the ensemble structures. The denatured-state ensemble and the early-stage folding were studied by a combination of unfolding simulations at 500 K and folding simulations at 300 K starting from the extended conformation. A common feature of the transition-state ensemble was the substantial formation of the native secondary structures, including both the alpha-helix and beta-sheet, with partial exposure of the hydrophobic core in the solvent. Both the native and non-native secondary structures were observed in the denatured-state ensemble and early-stage folding, consistent with the smooth experimental melting curve. Interestingly, the contact orders of the transition-state ensemble structures were similar to that of the native structure and were notably lower than those of the compact structures found in early-stage folding, implying that chain and topological entropy might play significant roles in protein folding. Implications for FSD-1 folding mechanisms and the rate-limiting step are discussed. Analyses further revealed interesting non-native interactions in the denatured-state ensemble and early-stage folding and the possibility that destabilization of these interactions could help to enhance the stability and folding rate of the protein.     

The driving force for co-translational protein folding is weaker in the ribosome vestibule due to greater water ordering

Interactions between the ribosome and nascent chain can destabilize folded domains in the ribosome exit tunnel''s vestibule, the last 3 nm of the exit tunnel where tertiary folding can occur. Here, we test if a contribution to this destabilization is a weakening of hydrophobic association, the driving force for protein folding. Using all-atom molecular dynamics simulations, we calculate the potential-of-mean force between two methane molecules along the center line of the ribosome exit tunnel and in bulk solution. Associated methanes, we find, are half as stable in the ribosome''s vestibule as compared to bulk solution, demonstrating that the hydrophobic effect is weakened by the presence of the ribosome. This decreased stability arises from a decrease in the amount of water entropy gained upon the association of the methanes. And this decreased entropy gain originates from water molecules being more ordered in the vestibule as compared to bulk solution. Therefore, the hydrophobic effect is weaker in the vestibule because waters released from the first solvation shell of methanes upon association do not gain as much entropy in the vestibule as they do upon release in bulk solution. These findings mean that nascent proteins pass through a ribosome vestibule environment that can destabilize folded structures, which has the potential to influence co-translational protein folding pathways, energetics, and kinetics.

In the ribosome vestibule, the contact minimum between two methane molecules is half as stable as compared to in bulk solution, demonstrating that the hydrophobic effect is weakened in the vestibule of ribosome exit tunnel.     

Relaxation rate for an ultrafast folding protein is independent of chemical denaturant concentration

The connection between free-energy surfaces and chevron plots has been investigated in a laser temperature jump kinetic study of a small ultrafast folding protein, the 35-residue subdomain from the villin headpiece. Unlike all other proteins that have been studied so far, no measurable dependence of the unfolding/refolding relaxation rate on denaturant concentration was observed over a wide range of guanidinium chloride concentration. Analysis with a simple Ising-like theoretical model shows that this denaturant-invariant relaxation rate can be explained by a large movement of the major free energy barrier, together with a denaturant- and reaction coordinate-dependent diffusion coefficient.     

磷光寿命法研究变性剂对大肠杆菌碱性磷酸酶构象的影响

色氨酸残基光寿命监测了大肠杆菌碱性磷酸酶在不同变性剂中展开过程的构象 变化．结果表明：不同变性剂加人蛋白质溶液中，色氨酸残基的微环境发生了较大 的变化，磷光发射减弱，寿命缩短，预示了色氨酸残基从刚性的疏水内芯转移到蛋 白质表面；通过Arrthenius关系式获得的热动力学参数如活化能（E_a）、活化熵 （△S°）、活开过程中间态的形成．     

Hydrophobic collapse in (in silico) protein folding

A model of hydrophobic collapse, which is treated as the driving force for protein folding, is presented. This model is the superposition of three models commonly used in protein structure prediction: (1) 'oil-drop' model introduced by Kauzmann, (2) a lattice model introduced to decrease the number of degrees of freedom for structural changes and (3) a model of the formation of hydrophobic core as a key feature in driving the folding of proteins. These three models together helped to develop the idea of a fuzzy-oil-drop as a model for an external force field of hydrophobic character mimicking the hydrophobicity-differentiated environment for hydrophobic collapse. All amino acids in the polypeptide interact pair-wise during the folding process (energy minimization procedure) and interact with the external hydrophobic force field defined by a three-dimensional Gaussian function. The value of the Gaussian function usually interpreted as a probability distribution is treated as a normalized hydrophobicity distribution, with its maximum in the center of the ellipsoid and decreasing proportionally with the distance versus the center. The fuzzy-oil-drop is elastic and changes its shape and size during the simulated folding procedure.     

Sampling of states for estimating the folding funnel entropy and energy landscape of a model alpha-helical hairpin peptide

Protein folding times are many orders of magnitude shorter than would occur if the peptide chain randomly sampled possible configurations, which implies that protein folding is a directed process. The detailed shape of protein's energy landscape determines the rate and reliability of folding to the native state, but the large number of structural degrees of freedom generates an energy landscape that is hard to visualize because of its high dimensionality. A commonly used picture is that of an energy funnel leading from high energy random coil state down to the low energy native state. As lattice computer models of protein dynamics become more realistic, the number of possible configurations becomes too large to count directly. Statistical mechanic and thermodynamic approaches allow us to count states in an approximate manner to quantify the entropy and energy of the energy landscape within a folding funnel for an alpha-helical protein. We also discuss the problems that arise in attempting to count the huge number of individual states of the random coil at the top of the funnel.