从高分子构象与动力学到蛋白质折叠

针对作用在聚合物刷上的键拉力研究表明作用在接枝基面上的力随着聚合物刷接枝密度的增大反而减小,然而尾端单体上的拉伸张力并没有消失.高分子的构象和动力学转变决定了其物性和多种多样的应用,而生物大分子蛋白质作为由二十种不同属性的氨基酸构成的序列,更是具有由其序列所决定的特别的三维自然结构.本文就聚合物刷、聚合物纳米复合材料、聚合物网络等几种高分子体系的构象与动力学过程,及蛋白质构象和其折叠与去折叠的动力学过程做了介绍.特别是蛋白质的折叠与去折叠速率在单分子操纵实验中受到拉力的调控,通过测量这种拉力依赖的动力学过程、蛋白质的自由能曲面和折叠去折叠路径可以得到系统全面的研究.本文以肌肉蛋白titin的免疫球蛋白结构域I27为例对蛋白质折叠研究进行了阐述.     

Multiple conformations of a protein demonstrated by magnetization transfer NMR spectroscopy

It is generally accepted that a globular protein in its native state adopts a single, well-defined conformation. However, there have been several reports that some proteins may exist in more than one distinct folded form in equilibrium. In the case of staphylococcal nuclease, evidence for multiple conformations has come from electrophoretic and NMR studies, although there has been some controversy as to whether these are actually interconvertible forms of the same molecular species. Recently, magnetization transfer (MT)-NMR has been developed as a means of studying the kinetics of conformational transitions in proteins. In the study reported here, this approach has been extended and used to demonstrate the presence of at least two native forms of nuclease in equilibrium and to study their interconversion with the unfolded state under the conditions of the thermal unfolding transition. The experiments reveal that two distinct native forms of the protein fold and unfold independently and that these can interconvert directly as well as via the unfolded state. The spectra of the different forms suggest that they are structurally similar but the MT experiments show that the kinetics of folding and unfolding are quite different. Characterization of this behaviour will, therefore, have important implications for our understanding of the relationship between structure and folding kinetics.     

Interpreting the folding kinetics of helical proteins.

The detailed mechanism of protein folding is one of the major problems in structural biology. Its solution is of practical as well as fundamental interest because of its possible role in utilizing the many sequences becoming available from genomic analysis. Although the Levinthal paradox (namely, that a polypeptide chain can find its unique native state in spite of the astronomical number of configurations in the denatured state) has been resolved, the reasons for the differences in the folding behaviour of individual proteins remain to be elucidated. Here a Calpha-based three-helix-bundle-like protein model with a realistic thermodynamic phase diagram is used to calculate several hundred folding trajectories. By varying a single parameter, the difference between the strength of native and non-native contacts, folding is changed from a diffusion-collision mechanism to one that involves simultaneous collapse and partial secondary-structure formation, followed by reorganization to the native structure. Non-obligatory intermediates are important in the former, whereas there is an obligatory on-pathway intermediate in the latter. Our results provide a basis for understanding the range of folding behaviour that is observed in helical proteins.     

The molten globule protein conformation probed by disulphide bonds

The molten globule is a compact protein conformation that has a secondary structure content like that of the native protein, but poorly defined tertiary structure. It is a stable state for a few proteins under particular conditions and could be a ubiquitous kinetic intermediate in protein folding. The extent to which native interactions, above the level of the secondary structure, are preserved in this conformation is not so far known. Here we report that alpha-lactalbumin can adopt a molten globule conformation when one of its four disulphide bonds is reduced. In this state, the three other disulphide bonds rearrange spontaneously, at the same rate as when the protein is fully unfolded, to a number of different disulphide bond isomers that tend to maintain the molten globule conformation. That the molten globule state is compatible with a variety of disulphide bond pairings suggests that it is unlikely to be stabilized by many specific tertiary interactions.     

Solution structure of a protein denatured state and folding intermediate

The most controversial area in protein folding concerns its earliest stages. Questions such as whether there are genuine folding intermediates, and whether the events at the earliest stages are just rearrangements of the denatured state or progress from populated transition states, remain unresolved. The problem is that there is a lack of experimental high-resolution structural information about early folding intermediates and denatured states under conditions that favour folding because competent states spontaneously fold rapidly. Here we have solved directly the solution structure of a true denatured state by nuclear magnetic resonance under conditions that would normally favour folding, and directly studied its equilibrium and kinetic behaviour. We engineered a mutant of Drosophila melanogaster Engrailed homeodomain that folds and unfolds reversibly just by changing ionic strength. At high ionic strength, the mutant L16A is an ultra-fast folding native protein, just like the wild-type protein; however, at physiological ionic strength it is denatured. The denatured state is a well-ordered folding intermediate, poised to fold by docking helices and breaking some non-native interactions. It unfolds relatively progressively with increasingly denaturing conditions, and so superficially resembles a denatured state with properties that vary with conditions. Such ill-defined unfolding is a common feature of early folding intermediate states and accounts for why there are so many controversies about intermediates versus compact denatured states in protein folding.     

The alpha-lytic protease pro-region does not require a physical linkage to activate the protease domain in vivo

alpha-Lytic protease, an extracellular serine protease of Lysobacter enzymogenes 495, is synthesized as a pre-pro-protein. Previously it has been shown that when expressed in Escherichia coli, the protein is autocatalytically processed in the periplasmic space, and that the functional protease domain accumulates extracellularly. Engineered proteins lacking the 166 amino-acid pro-region were enzymatically inactive and remained cell-associated. By independently expressing the pro- and protease domains in vivo, evidence is provided here that direct covalent linkage is not required for production of active protease. We postulate that the pro-region acts as a template to promote the folding of the protease domain into an active configuration. Our results, combined with recent experiments on the evolutionarily unrelated subtilisin E (ref. 3), suggest that the ability of the pro-region of these bacterial proteases to facilitate folding of their protease domains is not a curiosity of a single system, but may reflect a general property of extracellular bacterial serine proteases.     

Characterization of the Partially Folded Monomeric Intermediate of Creatine Kinase

IntroductionRecent studies of the protein folding pathway andintermediate states in vitro and in vivo haveinduced much interest in the importance ofunderstanding the propertiesof partially structuredintermediates[1 7] . Studies have suggested thatintermed…     

Molecular dynamics simulation of RNA pseudoknot unfolding pathway

Many biological functions of RNA molecules are related to their pseudoknot structures. It is significant for predicting the structure and function of RNA that learning about the stability and the process of RNA pseudoknot folding and unfolding. The structural features of mouse mammary tumor virus (MMTV) RNA pseudoknot in different ion concentration, the unfolding process of the RNA pseudoknot, and the two hairpin helices that constitute the RNA pseudoknot were studied with all atom molecule dynamics simulation method in this paper. We found that the higher cation concentration can cause structure of the RNA molecules more stable, and ions played an indispensable role in keeping the structure of RNA molecules stable; the unfolding process of hairpin structure was corresponding to the antiprocess of its folding process. The main pathway of pseudoknot unfolding was that the inner base pair opened first, and then, the two helices, which formed the RNA pseudoknot opened decussately, while the folding pathway of the RNA pseudoknot was a helix folding after formation of the other helix. Therefore, the unfolding process of RNA pseudoknot is different from the antiprocess of its folding process, and the unfolding process of each helix in the RNA pseudoknot is similar to the hairpin structure’s unfolding process, which means that both are the unzipping process.     

A 'molten-globule' membrane-insertion intermediate of the pore-forming domain of colicin A

The 'molten' globular conformation of a protein is compact with a native secondary structure but a poorly defined tertiary structure. Molten globular states are intermediates in protein folding and unfolding and they may be involved in the translocation or insertion of proteins into membranes. Here we investigate the membrane insertion of the pore-forming domain of colicin A, a bacteriocin that depolarizes the cytoplasmic membrane of sensitive cells. We find that this pore-forming domain, the insertion of which depends on pH, undergoes a native to molten globule transition at acidic pH. The variation of the kinetic constant of membrane insertion of the protein into negatively charged lipid vesicles as a function of the interfacial pH correlates with the appearance of the acidic molten globular state, indicating that this state could be an intermediate formed during the insertion of colicin A into membranes.     

Atom-by-atom analysis of global downhill protein folding

Protein folding is an inherently complex process involving coordination of the intricate networks of weak interactions that stabilize native three-dimensional structures. In the conventional paradigm, simple protein structures are assumed to fold in an all-or-none process that is inaccessible to experiment. Existing experimental methods therefore probe folding mechanisms indirectly. A widely used approach interprets changes in protein stability and/or folding kinetics, induced by engineered mutations, in terms of the structure of the native protein. In addition to limitations in connecting energetics with structure, mutational methods have significant experimental uncertainties and are unable to map complex networks of interactions. In contrast, analytical theory predicts small barriers to folding and the possibility of downhill folding. These theoretical predictions have been confirmed experimentally in recent years, including the observation of global downhill folding. However, a key remaining question is whether downhill folding can indeed lead to the high-resolution analysis of protein folding processes. Here we show, with the use of nuclear magnetic resonance (NMR), that the downhill protein BBL from Escherichia coli unfolds atom by atom starting from a defined three-dimensional structure. Thermal unfolding data on 158 backbone and side-chain protons out of a total of 204 provide a detailed view of the structural events during folding. This view confirms the statistical nature of folding, and exposes the interplay between hydrogen bonding, hydrophobic forces, backbone conformation and side-chain entropy. From the data we also obtain a map of the interaction network in this protein, which reveals the source of folding cooperativity. Our approach can be extended to other proteins with marginal barriers (less than 3RT), providing a new tool for the study of protein folding.     

Structure Characterization of the Folding Intermediates of Proteins

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A protein-folding reaction under kinetic control.

Synthesis of alpha-lytic protease is as a precursor containing a 166 amino-acid pro region transiently required for the correct folding of the protease domain. By omitting the pro region in an in vitro refolding reaction we trapped an inactive, but folding competent state (I) having an expanded radius yet native-like secondary structure. The I state is stable for weeks at physiological pH in the absence of denaturant, but rapidly folds to the active, native state on addition of the pro region as a separate polypeptide chain. The mechanism of action of the pro region is distinct from that of the chaperonins: rather than reducing the rate of off-pathway reactions, the pro region accelerates the rate-limiting step on the folding pathway by more than 10(7). Because both the I and native states are stable under identical conditions with no detectable interconversion, the folding of alpha-lytic protease must be under kinetic and not thermodynamic control.     

Implications of thermodynamics of protein folding for evolution of primary sequences

Natural proteins exhibit essentially two-state thermodynamics, with one stable fold that dominates thermodynamically over a vast number of possible folds, a number that increases exponentially with the size of the protein. Here we address the question of whether this feature of proteins is a rare property selected by evolution or whether it is in fact true of a significant proportion of all possible protein sequences. Using statistical procedures developed to study spin glasses, we show that, given certain assumptions, the probability that a randomly synthesized protein chain will have a dominant fold (which is the global minimum of free energy) is a function of temperature, and that below a critical temperature the probability rapidly increases as the temperature decreases. Our results suggest that a significant proportion of all possible protein sequences could have a thermodynamically dominant fold.     

Three key residues form a critical contact network in a protein folding transition state

Determining how a protein folds is a central problem in structural biology. The rate of folding of many proteins is determined by the transition state, so that a knowledge of its structure is essential for understanding the protein folding reaction. Here we use mutation measurements--which determine the role of individual residues in stabilizing the transition state--as restraints in a Monte Carlo sampling procedure to determine the ensemble of structures that make up the transition state. We apply this approach to the experimental data for the 98-residue protein acylphosphatase, and obtain a transition-state ensemble with the native-state topology and an average root-mean-square deviation of 6 A from the native structure. Although about 20 residues with small positional fluctuations form the structural core of this transition state, the native-like contact network of only three of these residues is sufficient to determine the overall fold of the protein. This result reveals how a nucleation mechanism involving a small number of key residues can lead to folding of a polypeptide chain to its unique native-state structure.     

Single-molecule fluorescence reveals sequence-specific misfolding in multidomain proteins

A large range of debilitating medical conditions is linked to protein misfolding, which may compete with productive folding particularly in proteins containing multiple domains. Seventy-five per cent of the eukaryotic proteome consists of multidomain proteins, yet it is not understood how interdomain misfolding is avoided. It has been proposed that maintaining low sequence identity between covalently linked domains is a mechanism to avoid misfolding. Here we use single-molecule F?rster resonance energy transfer to detect and quantify rare misfolding events in tandem immunoglobulin domains from the I band of titin under native conditions. About 5.5 per cent of molecules with identical domains misfold during refolding in vitro and form an unexpectedly stable state with an unfolding half-time of several days. Tandem arrays of immunoglobulin-like domains in humans show significantly lower sequence identity between neighbouring domains than between non-adjacent domains. In particular, the sequence identity of neighbouring domains has been found to be preferentially below 40 per cent. We observe no misfolding for a tandem of naturally neighbouring domains with low sequence identity (24 per cent), whereas misfolding occurs between domains that are 42 per cent identical. Coarse-grained molecular simulations predict the formation of domain-swapped structures that are in excellent agreement with the observed transfer efficiency of the misfolded species. We infer that the interactions underlying misfolding are very specific and result in a sequence-specific domain-swapping mechanism. Diversifying the sequence between neighbouring domains seems to be a successful evolutionary strategy to avoid misfolding in multidomain proteins.     

Hsp90 chaperones protein folding in vitro.

The heat-shock protein Hsp90 is the most abundant constitutively expressed stress protein in the cytosol of eukaryotic cells, where it participates in the maturation of other proteins, modulation of protein activity in the case of hormone-free steroid receptors, and intracellular transport of some newly synthesized kinases. A feature of all these processes could be their dependence on the formation of protein structure. If Hsp90 is a molecular chaperone involved in maintaining a certain subset of cellular proteins in an inactive form, it should also be able to recognize and bind non-native proteins, thereby influencing their folding to the native state. Here we investigate whether Hsp90 can influence protein folding in vitro and show that Hsp90 suppresses the formation of protein aggregates by binding to the target proteins at a stoichiometry of one Hsp90 dimer to one or two substrate molecule(s). Furthermore, the yield of correctly folded and functional protein is increased significantly. The action of Hsp90 does not depend on the presence of nucleoside triphosphates, so it may be that Hsp90 uses a novel molecular mechanism to assist protein folding in vivo.     

A pre-existing hydrophobic collapse in the unfolded state of an ultrafast folding protein

Insights into the conformational passage of a polypeptide chain across its free energy landscape have come from the judicious combination of experimental studies and computer simulations. Even though some unfolded and partially folded proteins are now known to possess biological function or to be involved in aggregation phenomena associated with disease states, experimentally derived atomic-level information on these structures remains sparse as a result of conformational heterogeneity and dynamics. Here we present a technique that can provide such information. Using a 'Trp-cage' miniprotein known as TC5b (ref. 5), we report photochemically induced dynamic nuclear polarization NMR pulse-labelling experiments that involve rapid in situ protein refolding. These experiments allow dipolar cross-relaxation with hyperpolarized aromatic side chain nuclei in the unfolded state to be identified and quantified in the resulting folded-state spectrum. We find that there is residual structure due to hydrophobic collapse in the unfolded state of this small protein, with strong inter-residue contacts between side chains that are relatively distant from one another in the native state. Prior structuring, even with the formation of non-native rather than native contacts, may be a feature associated with fast folding events in proteins.     

Demonstration by NMR of folding domains in lysozyme

Although there has been much speculation on the pathways of protein folding, only recently have experimental data on the topic been available. The study of proteins under conditions where species intermediate between the fully folded and unfolded states are stable has provided important information, for example about the disulphide intermediates in BPTI, cis/trans proline isomers of RNase A3 and the molten globule state of alpha-lactalbumin. An alternative approach to investigating folding pathways has involved detection and characterization of transient conformers in refolding studies using stopped-flow methods coupled with NMR measurements of hydrogen exchange. The formation of intermediate structures has been detected in the early stages of folding of cytochrome c, RNaseA and barnase. For alpha-lactalbumin, hydrogen exchange kinetics monitored by NMR proved to be crucial for identifying native-like structural features in the stable molten globule state. An analogous partially folded protein stable under equilibrium conditions has not been observed for the structurally homologous protein hen egg-white lysozyme, although there is evidence that a similar but transient state is formed during refolding. Here we describe NMR experiments based on competition between hydrogen exchange and the refolding process which not only support the existence of such a transient species for lysozyme, but enable its structural characteristics to be defined. The results indicate that the two structural domains of lysozyme are distinct folding domains, in that they differ significantly in the extent to which compact, probably native-like, structure is present in the early stages of folding.     

Role of a subdomain in the folding of bovine pancreatic trypsin inhibitor.

The disulphide-bonded intermediates that accumulate in the oxidative folding of bovine pancreatic trypsin inhibitor (BPTI) were characterized some time ago. Structural characterization of these intermediates would provide an explanation of the kinetically preferred pathways of folding for BPTI. When folding occurs under strongly oxidizing conditions, more than half the molecules become trapped in an intermediate, designated N*, which is similar to the native protein but lacks the 30-51 disulphide bond. We have tested the hypothesis that the precursor to N* is the one-disulphide intermediate [5-55], which contains the most stable disulphide in BPTI, and present evidence here that this is the case. A peptide model of [5-55], corresponding to a subdomain of BPTI, seems to fold into a native-like conformation, explaining why [5-55] does not lead to native protein and why it folds rapidly to N*. A native-like subdomain structure in a peptide model of [30-51], the other crucial one-disulphide intermediate, may explain the route by which [30-51] folds to native protein. Thus, much of the folding pathway of BPTI can be explained by the formation of a native-like subdomain in these two early intermediates. This suggests that a large part of the protein folding problem can be reduced to identifying and understanding subdomains of native proteins.