Protein dynamics: From the native to the unfolded state and back again

Simulations to study protein unfolding and folding were performed. The unfolding simulations make use of molecular dynamics and treat an atomic model of barnase in aqueous solvent. The cooperative nature of the unfolding transition and the important role of water are described. The folding simulations are based on a bead model of the protein on a cubic lattice. It is shown for the 27-mer model that a large energy gap between the lowest energy (native) state and the excited states is a necessary and sufficient condition for fast folding.     

Exploring the role of hydrophilic amino acids in unfolding of protein in aqueous ethanol solution

Hydrophobic association is the key contributor behind the formation of well packed core of a protein which is often believed to be an important step for folding from an unfolded chain to its compact functional form. While most of the protein folding/unfolding studies have evaluated the changes in the hydrophobic interactions during chemical denaturation, the role of hydrophilic amino acids in such processes are not discussed in detail. Here we report the role of the hydrophilic amino acids behind ethanol induced unfolding of protein. Using free energy simulations, we show that chicken villin head piece (HP‐36) protein unfolds gradually in presence of water‐ethanol binary mixture with increasing composition of ethanol. However, upon mutation of hydrophilic amino acids by glycine while keeping the hydrophobic amino acids intact, the compact state of the protein is found to be stable at all compositions with gradual flattening of the free energy landscape upon increasing compositions. The local environment around the protein in terms of ethanol/water number significantly differs in wild type protein compared to the mutated protein. The calculated Wyman‐Tanford preferential binding coefficient of ethanol for wild type protein reveals that a greater number of cosolutes (here ethanol) bind to the unfolded state compared to its folded state. However, no significant increase in binding coefficient of ethanol at the unfolded state is found for mutated protein. Local‐bulk partition coefficient calculation also suggests similar scenarios. Our results reveal that the weakening of hydrophobic interactions in aqueous ethanol solution along with larger preferential binding of ethanol to the unfolded state mediated by hydrophilic amino acids combinedly helps unfolding of protein in aqueous ethanol solution.     

未折叠蛋白质的普遍初始热力学亚稳态

探索和理解蛋白质折叠问题一直是分子生物学、结构生物学和生物物理学的终极挑战.未折叠的蛋白质应该存在一种普遍初始热力学亚稳态,否则无法解释蛋白质是如何在剧烈的热振动干扰下完成快速精确折叠的.本文通过分析水溶液环境和蛋白质折叠的相关性,揭示了一种由水分子屏蔽效应引起的未折叠蛋白质的普遍初始热力学亚稳态,该亚稳态的存在是水溶液环境中水分子的物理性质决定,并赋予未折叠蛋白质抵抗热扰动和避免错误折叠的能力.我们通过研究已发表的实验数据和建立分子模型,找到了该初始热力学亚稳态存在的相关证据,并推测了该亚稳态导致蛋白质精确折叠的相关物理学机制.     

Is an intermediate state populated on the folding pathway of ubiquitin?

In the last couple of years, there has been increasing debate as to the presence and role of intermediate states on the folding pathways of several small proteins, including the 76-residue protein ubiquitin. Here, we present detailed kinetic studies to establish whether an intermediate state is ever populated during the folding of this protein. We show that the differences observed in previous studies are attributable to the transient aggregation of the protein during folding. Using a highly soluble construct of ubiquitin, which does not aggregate during folding, we establish the conditions in which an intermediate state is sufficiently stable to be observed by kinetic measurements.     

Effects of confinement in chaperonin assisted protein folding: rate enhancement by decreasing the roughness of the folding energy landscape

Chaperonins, such as the GroE complex of the bacteria Escherichia coli, assist the folding of proteins under non-permissive folding conditions by providing a cavity in which the newly translated or translocated protein can be encapsulated. Whether the chaperonin cage plays a passive role in protecting the protein from aggregation, or an active role in accelerating folding rates, remains a matter of debate. Here, we investigate the role of confinement in chaperonin mediated folding through molecular dynamics simulations. We designed a substrate protein with an alpha/beta sandwich fold, a common structural motif found in GroE substrate proteins and confined it to a spherical hydrophilic cage which mimicked the interior of the GroEL/ES cavity. The thermodynamics and kinetics of folding were studied over a wide range of temperature and cage radii. Confinement was seen to significantly raise the collapse temperature, T(c), as a result of the associated entropy loss of the unfolded state. The folding temperature, T(f), on the other hand, remained unaffected by encapsulation, a consequence of the folding mechanism of this protein that involves an initial collapse to a compact misfolded state prior to rearranging to the native state. Folding rates were observed to be either accelerated or retarded compared to bulk folding rates, depending on the temperature of the simulation. Rate enhancements due to confinement were observed only at temperatures above the temperature T(m), which corresponds to the temperature at which the protein folds fastest. For this protein, T(m) lies above the folding temperature, T(f), implying that encapsulation alone will not lead to a rate enhancement under conditions where the native state is stable (T相似文献   

Ultrafast Protein Folding in Membrane-Mimetic Environments

Proteins fold on timescales from hours to microseconds. In addition to protein size, sequence, and topology, the environment represents an equally important factor in determining folding speed. This is particularly relevant for proteins that require a lipid membrane or a membrane mimic to fold. However, only little is known about how properties of such a hydrophilic/hydrophobic interface modulate the folding landscape of membrane-interacting proteins. Here, we studied the influence of different membrane-mimetic micellar environments on the folding and unfolding kinetics of the helical-bundle protein Mistic. Devising a single-molecule fluorescence spectroscopy approach, we extracted folding and unfolding rates under equilibrium conditions and dissected the contributions from different detergent moieties to the free-energy landscape. While both polar and nonpolar moieties contribute to stability, they exert differential effects on the free-energy barrier: Hydrophobic burial stabilizes the folded state but not the transition state in reference to a purely aqueous environment; by contrast, zwitterionic headgroup moieties stabilize the folded state and, additionally, lower the free-energy barrier to accelerate the folding of Mistic to achieve ultrafast folding times down to 35 μs.     

Nonnative Electrostatic Interactions Can Modulate Protein Folding: Molecular Dynamics with a Grain of Salt

In recent years, a growing number of protein folding studies have focused on the unfolded state, which is now recognized as playing a major role in the folding process. Some of these studies show that interactions occurring in the unfolded state can significantly affect the stability and kinetics of the protein folding reaction. In this study, we modeled the effect of electrostatic interactions, both native and nonnative, on the folding of three protein systems that underwent selective charge neutralization or reversal or complete charge suppression. In the case of the N-terminal L9 protein domain, our results directly attribute the increase in thermodynamic stability to destabilization of the unfolded ensemble, reaffirming the experimental observations. These results provide a deeper structural insight into the ensemble of the unfolded state and predict a new mutation site for increased protein stability. In the second case, charge reversal mutations of RNase Sa affected protein stability, with the destabilizing mutations being less destabilizing at higher salt concentrations, indicating the formation of charge-charge interactions in the unfolded state. In the N-terminal L9 and RNase Sa systems, changes in electrostatic interactions in the unfolded state that cause an increase in free energy had an overall compaction effect that suggests a decrease in entropy. In the third case, in which we compared the β-lactalbumin and hen egg-white lysozyme protein homologues, we successfully eliminated differences between the folding kinetics of the two systems by suppressing electrostatic interactions, supporting previously reported findings. Our coarse-grained molecular dynamics study not only reproduces experimentally reported findings but also provides a detailed molecular understanding of the elusive unfolded-state ensemble and how charge-charge interactions can modulate the biophysical characteristics of folding.     

Molecular thermodynamic model to predict the alpha-helical secondary structure of polypeptide chains in solution.

The native state of a protein molecule in aqueous solutions represents one of the lowest states of Gibbs energy [Anfinsen, C.B. (1973) Science 181, 223-230]. Much progress has been made about the rules of protein folding [King, J. (1989) Chem. Eng. News 67, 32-54] and the dominant forces in protein folding [Dill, K.A. (1990) Biochemistry 29, 7133-7155]. However, the quantitative contributions of different Gibbs energy terms to protein stability remains a controversial issue [Moult, J., & Unger, R. (1991) Biochemistry 30, 3816-3824]. A molecular thermodynamic model has been proposed for the Gibbs energy of folding a residue in aqueous homopolypeptides from a random-coiled state to either the alpha-helix state or the beta-sheet state [Chen, C.-C., Zhu, Y., King, J.A., & Evans, L.B. (1992) Biopolymers 32, 1375-1392]. In this work, we present a generalization of the molecular thermodynamic model for the Gibbs energy of folding natural and synthetic heteropolypeptides from random-coiled conformations into alpha-helical conformations. The generalized model incorporates the intrinsic folding potential due to residue-solvent interactions, the cooperative folding effect due to residue-residue interactions, and the location and length of alpha-helices. The utility of the model was demonstrated by examining the stability of alpha-helical conformations of a number of natural polypeptides including C-peptide (residues 1-13) and S-peptide (residues 1-20) of RNase A (bovine pancreatic ribonuclease A), the P alpha fragment in BPTI (bovine pancreatic trypsin inhibitor), and synthetic polypeptides (the copolymers of different amino acid residues) including alanine-based peptides (16 or 17 residues long) in water. The computed Gibbs energies correspond well with the experimental data on helicity. The results also accounted for the effects of amino acid substitution and temperature on the stability of alpha-helical conformations of the test polypeptides.     

Ubiquitin folds via a flip-twist-lock mechanism

To perform specific functional activities, the majority of proteins should fold into their distinct three-dimensional conformations. However, the biologically active conformation of a protein is generally found to be marginally stable than the other conformations that the chain can adopt. How a protein finds its native conformation from its post-synthesis unfolded structure in a complex conformational landscape is the unsolved question that still drives the protein folding community. Here, we report the folding mechanism of a globular protein, ubiquitin, from its chemically denatured state using all-atom molecular dynamics simulations. From the kinetic analysis of the simulated trajectories we show that the folding process can be described by the hydrophobic collapse mechanism, initiated by the “dewetting transition”, and subsequently assisted by the origination of an N-terminal folding nucleus, and finally supported by a native salt-bridge interaction between K11 and E34. We show that ubiquitin folds via an intermediate. Finally, we confirm the presence of “biological water” and explain its role to the folding process.     

The brace for a growing scaffold: mss116 protein promotes RNA folding by stabilizing an early assembly intermediate

The ai5γ group II intron requires a protein cofactor to facilitate native folding in the cell. Yeast protein Mss116 greatly accelerates intron folding under near-physiological conditions both in vivo and in vitro. Although the effect of Mss116 on the kinetics of ai5γ ribozyme folding and catalysis has been extensively studied, the precise structural role and interaction sites of Mss116 have been elusive. Using Nucleotide Analog Interference Mapping to study the folding of splicing precursor constructs, we have identified specific intron functional groups that participate in Mss116-facilitated folding and we have determined their role in the folding mechanism. The data indicate that Mss116 stabilizes an early, obligate folding intermediate within intron domain 1, thereby laying the foundation for productive folding to the native state. In addition, the data reveal an important role for the IBS2 exon sequence and for the terminus of domain 6, during the folding of self-splicing group IIB intron constructs.     

Unfolded state of polyalanine is a segmented polyproline II helix

Definition of the unfolded state of proteins is essential for understanding their stability and folding on biological timescales. Here, we find that under near physiological conditions the configurational ensemble of the unfolded state of the simplest protein structure, polyalanine alpha-helix, cannot be described by the commonly used Flory random coil model, in which configurational probabilities are derived from conformational preferences of individual residues. We utilize novel effectively ergodic sampling algorithms in the presence of explicit aqueous solvation, and observe water-mediated formation of polyproline II helical (P(II)) structure in the natively unfolded state of polyalanine, and its facilitation of alpha-helix formation in longer peptides. The segmented P(II) helical coil preorganizes the unfolded state ensemble for folding pathway entry by reducing the conformational space available to the diffusive search. Thus, as much as half of the folding search in polyalanine is accomplished by preorganization of the unfolded state.     

Microscopic reversibility of protein folding in molecular dynamics simulations of the engrailed homeodomain

The principle of microscopic reversibility states that at equilibrium the number of molecules entering a state by a given path must equal those exiting the state via the same path under identical conditions or, in structural terms, that the conformations along the two pathways are the same. There has been some indirect evidence indicating that protein folding is such a process, but there have been few conclusive findings. In this study, we performed molecular dynamics simulations of an ultrafast unfolding and folding protein at its melting temperature to observe, on an atom-by-atom basis, the pathways the protein followed as it unfolded and folded within a continuous trajectory. In a total of 0.67 micros of simulation in water, we found six transient denaturing events near the melting temperature (323 and 330 K) and an additional refolding event following a previously identified unfolding event at a high temperature (373 K). In each case, unfolding and refolding transition state ensembles were identified, and they agreed well with experiment on the basis of a comparison of S and Phi values. On the basis of several structural properties, these 13 transition state ensembles agreed very well with each other and with four previously identified transition states from high-temperature denaturing simulations. Thus, not only were the unfolding and refolding transition states part of the same ensemble, but in five of the seven cases, the pathway the protein took as it unfolded was nearly identical to the subsequent refolding pathway. These events provide compelling evidence that protein folding is a microscopically reversible process. In the other two cases, the folding and unfolding transition states were remarkably similar to each other but the paths deviated.     

Folding propensities of peptide fragments of myoglobin.

Myoglobin has been studied extensively as a paradigm for protein folding. As part of an ongoing study of potential folding initiation sites in myoglobin, we have synthetized a series of peptides covering the entire sequence of sperm whale myoglobin. We report here on the conformation preferences of a series of peptides that cover the region from the A helix to the FG turn. Structural propensities were determined using circular dichroism and nuclear magnetic resonance spectroscopy in aqueous solution, trifluoroethanol, and methanol. Peptides corresponding to helical regions in the native protein, namely the B, C, D, and E helices, populate the alpha region of (phi, psi) space in water solution but show no measurable helix formation except in the presence of trifluoroethanol. The F-helix sequence has a much lower propensity to populate helical conformations even in TFE. Despite several attempts, we were not successful in synthesizing a peptide corresponding to the A-helix region that was soluble in water. A peptide termed the AB domain was constructed spanning the A- and B-helix sequences. The AB domain is not soluble in water, but shows extensive helix formation throughout the peptide when dissolved in methanol, with a break in the helix at a site close to the A-B helix junction in the intact folded myoglobin protein. With the exception of one local preference for a turn conformation stabilized by hydrophobic interactions, the peptides corresponding to turns in the folded protein do not measurably populate beta-turn conformations in water, and the addition of trifluoroethanol does not enhance the formation of either helical or turn structure. In contrast to the series of peptides described here, either studies of peptides from the GH region of myoglobin show a marked tendency to populate helical structures (H), nascent helical structures (G), or turn conformations (GH peptide) in water solution. This region, together with the A-helix and part of the B-helix, has been shown to participate in an early folding intermediate. The complete analysis of conformational properties of isolated myoglobin peptides supports the hypothesis that spontaneous secondary structure formation in local regions of the polypeptide may play an important role in the initiation of protein folding.     

Does native state topology determine the RNA folding mechanism?

Recent studies in protein folding suggest that native state topology plays a dominant role in determining the folding mechanism, yet an analogous statement has not been made for RNA, most likely due to the strong coupling between the ionic environment and conformational energetics that make RNA folding more complex than protein folding. Applying a distributed computing architecture to sample nearly 5000 complete tRNA folding events using a minimalist, atomistic model, we have characterized the role of native topology in tRNA folding dynamics: the simulated bulk folding behavior predicts well the experimentally observed folding mechanism. In contrast, single-molecule folding events display multiple discrete folding transitions and compose a largely diverse, heterogeneous dynamic ensemble. This both supports an emerging view of heterogeneous folding dynamics at the microscopic level and highlights the need for single-molecule experiments and both single-molecule and bulk simulations in interpreting bulk experimental measurements.     

Protein translocation through a tunnel induces changes in folding kinetics: a lattice model study

Compaction of a nascent polypeptide chain inside the ribosomal exit tunnel, before it leaves the ribosome, has been proposed to accelerate the folding of newly synthesized proteins following their release from the ribosome. Thus, we used Kinetic Monte Carlo simulations of a minimalist on-lattice model to explore the effect that polypeptide translocation through a variety of channels has on protein folding kinetics. Our results demonstrate that tunnel confinement promotes faster folding of a well-designed protein relative to its folding in free space by displacing the unfolded state towards more compact structures that are closer to the transition state. Since the tunnel only forbids rarely visited, extended configurations, it has little effect on a "poorly designed" protein whose unfolded state is largely composed of low-energy, compact, misfolded configurations. The beneficial effect of the tunnel depends on its width; for example, a too-narrow tunnel enforces unfolded states with limited or no access to the transition state, while a too-wide tunnel has no effect on the unfolded state entropy. We speculate that such effects are likely to play an important role in the folding of some proteins or protein domains in the cellular environment and might dictate whether a protein folds co-translationally or post-translationally.     

Parallel tempering molecular dynamics folding simulation of a signal peptide in explicit water

Parallel temperature molecular dynamics simulations are used to explore the folding of a signal peptide, a short but functionally independent domain at the N-terminus of proteins. The peptide has been analyzed previously by NMR, and thus a solid reference state is provided with the experimental structure. Particular attention is paid to the role of water considered in full atomic detail. Different partial aspects in the folding process are quantified. The major group of obtained structures matches the NMR structure very closely. An important biological consequence is that in vivo folding of signal peptides seems to be possible within aqueous environments.     

Proline inhibits aggregation during protein refolding

The in vitro refolding of hen egg-white lysozyme is studied in the presence of various osmolytes. Proline is found to prevent aggregation during protein refolding. However, other osmolytes used in this study fail to exhibit a similar property. Experimental evidence suggests that proline inhibits protein aggregation by binding to folding intermediate(s) and trapping the folding intermediate(s) into enzymatically inactive, "aggregation-insensitive" state(s). However, elimination of proline from the refolded protein mixture results in significant recovery of the bacteriolytic activity. At higher concentrations (>1.5 M), proline is shown to form loose, higher-order molecular aggregate(s). The supramolecular assembly of proline is found to possess an amphipathic character. Formation of higher-order aggregates is believed to be crucial for proline to function as a protein folding aid. In addition to its role in osmoregulation under water stress conditions, the results of this study hint at the possibility of proline behaving as a protein folding chaperone.     

Heat capacity of hydrogen-bonded networks: an alternative view of protein folding thermodynamics

Large changes in heat capacity (deltaCp) have long been regarded as the characteristic thermodynamic signature of hydrophobic interactions. However, similar effects arise quite generally in order-disorder transitions in homogeneous systems, particularly those comprising hydrogen-bonded networks, and this may have significance for our understanding of protein folding and other biomolecular processes. The positive deltaCp associated with unfolding of globular proteins in water, thought to be due to hydrophobic interactions, is also typical of the values found for the melting of crystalline solids, where the effect is greatest for the melting of polar compounds, including pure water. This suggests an alternative model of protein folding based on the thermodynamics of phase transitions in hydrogen-bonded networks. Folded proteins may be viewed as islands of cooperatively-ordered hydrogen-bonded structure, floating in an aqueous network of less-well-ordered H-bonds in which the degree of hydrogen bonding decreases with increasing temperature. The enthalpy of melting of the protein consequently increases with temperature. A simple algebraic model, based on the overall number of protein and solvent hydrogen bonds in folded and unfolded states, shows how deltaCp from this source could match the hydrophobic contribution. This confirms the growing view that the thermodynamics of protein folding, and other interactions in aqueous systems, are best described in terms of a mixture of polar and non-polar effects in which no one contribution is necessarily dominant.     

Solvation of the folding-transition state in Pseudomonas aeruginosa azurin is modulated by metal: Solvation of azurin's folding nucleus

The role of water in protein folding, specifically its presence or not in the transition-state structure, is an unsolved question. There are two common classes of folding-transition states: diffuse transition states, in which almost all side chains have similar, rather low phi (phi) values, and polarized transition states, which instead display distinct substructures with very high phi-values. Apo-and zinc-forms of Pseudomonas aeruginosa azurin both fold in two-state equilibrium and kinetic reactions; while the apo-form exhibits a polarized transition state, the zinc form entails a diffuse, moving transition state. To examine the presence of water in these two types of folding-transition states, we probed the equilibrium and kinetic consequences of replacing core valines with isosteric threonines at six positions in azurin. In contrast to regular hydrophobic-to-alanine phi-value analysis, valine-to-threonine mutations do not disrupt the core packing but stabilize the unfolded state and can be used to assess the degree of solvation in the folding-transition state upon combination with regular phi-values. We find that the transition state for folding of apo-azurin appears completely dry, while that for zinc-azurin involves partially formed interactions that engage water molecules. This distinct difference between the apo-and holo-folding nuclei can be rationalized in terms of the shape of the free-energy barrier.