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.     

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.     

Rationalization of the effects of mutations on peptide and protein aggregation rates

In order for any biological system to function effectively, it is essential to avoid the inherent tendency of proteins to aggregate and form potentially harmful deposits. In each of the various pathological conditions associated with protein deposition, such as Alzheimer's and Parkinson's diseases, a specific peptide or protein that is normally soluble is deposited as insoluble aggregates generally referred to as amyloid. It is clear that the aggregation process is generally initiated from partially or completely unfolded forms of the peptides and proteins associated with each disease. Here we show that the intrinsic effects of specific mutations on the rates of aggregation of unfolded polypeptide chains can be correlated to a remarkable extent with changes in simple physicochemical properties such as hydrophobicity, secondary structure propensity and charge. This approach allows the pathogenic effects of mutations associated with known familial forms of protein deposition diseases to be rationalized, and more generally enables prediction of the effects of mutations on the aggregation propensity of any polypeptide chain.     

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.     

Successive action of DnaK, DnaJ and GroEL along the pathway of chaperone-mediated protein folding.

The main stress proteins of Escherichia coli function in an ordered protein-folding reaction. DnaK (heat-shock protein 70) recognizes the folding polypeptide as an extended chain and cooperates with DnaJ in stabilizing an intermediate conformational state lacking ordered tertiary structure. Dependent on GrpE and ATP hydrolysis, the protein is then transferred to GroEL (heat-shock protein 60) which acts catalytically in the production of the native state. This sequential mechanism of chaperone action may represent an important pathway for the folding of newly synthesized polypeptides.     

Structure Characterization of the Folding Intermediates of Proteins

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Catalysis of protein folding by prolyl isomerase

Rates of protein folding reactions vary considerably. Some denatured proteins regain the native conformation within milliseconds or seconds, whereas others refold very slowly in the time range of minutes or hours. Varying folding rates are observed not only for different proteins, but can also be detected for single polypeptide species. This originates from the co-existence of fast- and slow-folding forms of the unfolded protein, which regain the native state with different rates. The proline hypothesis provides a plausible explanation for this heterogeneity. It assumes that the slow-folding molecules possess non-native isomers of peptide bonds between proline and another residue, and that crucial steps in the refolding of the slow-folding molecules are limited in rate by the slow reisomerization of such incorrect proline peptide bonds. Recently the enzyme peptidyl-prolyl cis-trans isomerase (PPIase) was discovered and purified from pig kidney. It catalyses efficiently the cis in equilibrium trans isomerization of proline imidic peptide bonds in oligopeptides. Here we show that it also catalyses slow steps in the refolding of a number of proteins of which fast- and slow-folding species have been observed and where it was suggested that proline isomerization was involved in slow refolding. The efficiency of catalysis depends on the accessibility for the isomerase of the particular proline peptide bonds in the refolding protein chain.     

Principles of Quantitative Estimation of the Chaperone-Like Activity

IntroductionThe folding of most newly synthesized proteins inthe cell requires the interaction of a variety ofprotein cofactors known as molecular chaperones.These molecules recognize and bind to nascentpolypeptide chains and partially foldedintermediates of proteins,preventing theiraggregation and misfolding[1 7] .　 There are several classes of chaperones definedusing molecular size,cellular compartment andfunction. Thelargestchaperonefamilies are Hsp90(heat shock protein of molecular mass …     

Reverse hydrophobic effects relieved by amino-acid substitutions at a protein surface

It is rare for amino-acid substitutions on the surface of proteins to have large stabilizing or destabilizing effects. Nevertheless, one substitution of this type, the Tyr 26----Cys mutation in lambda Cro, increases the melting temperature of the protein by 11 degrees C and the stability by 2.2 kcal mol-1. Here we show that the stability of Cro can be increased by many different amino-acid substitutions at position 26, with increasing stability showing a good correlation with decreasing side-chain hydrophobicity. As Tyr 26 is hyper-exposed to solvent in the Cro crystal structure, we suggest that wild-type and variant proteins with other hydrophobic side chains at position 26 are destabilized as a result of a reverse hydrophobic effect caused by the side chain being more exposed to solvent in the native than in the denatured state.     

Simultaneous determination of protein structure and dynamics

We present a protocol for the experimental determination of ensembles of protein conformations that represent simultaneously the native structure and its associated dynamics. The procedure combines the strengths of nuclear magnetic resonance spectroscopy--for obtaining experimental information at the atomic level about the structural and dynamical features of proteins--with the ability of molecular dynamics simulations to explore a wide range of protein conformations. We illustrate the method for human ubiquitin in solution and find that there is considerable conformational heterogeneity throughout the protein structure. The interior atoms of the protein are tightly packed in each individual conformation that contributes to the ensemble but their overall behaviour can be described as having a significant degree of liquid-like character. The protocol is completely general and should lead to significant advances in our ability to understand and utilize the structures of native proteins.     

Transient association of newly synthesized unfolded proteins with the heat-shock GroEL protein

It has been suggested that newly synthesized proteins are maintained in their unfolded state by cellular ATP-driven factors which may prevent or reverse the formation of misfolded structures or promote the correct assembly of oligomeric proteins or post-translational secretion. Using a photocross-linking approach, we have identified the 20S heat-shock GroEL protein as the major cytosolic component which forms a complex with the unfolded newly synthesized pre-beta-lactamase or chloramphenicol acetyltransferase in Escherichia coli. Dissociation of these complexes is ATP-dependent. The unfolded state of pre-beta-lactamase, maintained by the transient interaction with GroEL, may be essential for the secretion of this protein.     

Visualization of release factor 3 on the ribosome during termination of protein synthesis

Termination of protein synthesis by the ribosome requires two release factor (RF) classes. The class II RF3 is a GTPase that removes class I RFs (RF1 or RF2) from the ribosome after release of the nascent polypeptide. RF3 in the GDP state binds to the ribosomal class I RF complex, followed by an exchange of GDP for GTP and release of the class I RF. As GTP hydrolysis triggers release of RF3 (ref. 4), we trapped RF3 on Escherichia coli ribosomes using a nonhydrolysable GTP analogue. Here we show by cryo-electron microscopy that the complex can adopt two different conformational states. In 'state 1', RF3 is pre-bound to the ribosome, whereas in 'state 2' RF3 contacts the ribosome GTPase centre. The transfer RNA molecule translocates from the peptidyl site in state 1 to the exit site in state 2. This translocation is associated with a large conformational rearrangement of the ribosome. Because state 1 seems able to accommodate simultaneously both RF3 and RF2, whose position is known from previous studies, we can infer the release mechanism of class I RFs.     

Substantial increase of protein stability by multiple disulphide bonds

Disulphide bonds can significantly stabilize the native structures of proteins. The effect is presumed to be due mainly to a decrease in the configurational chain entropy of the unfolded polypeptide. In phage T4 lysozyme, a disulphide-free enzyme, engineered disulphide mutants that crosslink residues 3-97, 9-164 and 21-142 are significantly more stable than the wild-type protein. To investigate the effect of multiple-disulphide bonds on protein stability, mutants were constructed in which two or three stabilizing disulphide bridges were combined in the same protein. Reversible thermal denaturation shows that the increase in melting temperature resulting from the individual disulphide bonds is approximately additive. The triple-disulphide variant unfolds at a temperature 23.4 degrees C higher than wild-type lysozyme. The results demonstrate that a combination of disulphide bonds, each of which contributes to stability, can achieve substantial overall improvement in the stability of a protein.     

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.     

Structural insights into a yeast prion illuminate nucleation and strain diversity

Self-perpetuating changes in the conformations of amyloidogenic proteins play vital roles in normal biology and disease. Despite intense research, the architecture and conformational conversion of amyloids remain poorly understood. Amyloid conformers of Sup35 are the molecular embodiment of the yeast prion known as [PSI], which produces heritable changes in phenotype through self-perpetuating changes in protein folding. Here we determine the nature of Sup35's cooperatively folded amyloid core, and use this information to investigate central questions in prion biology. Specific segments of the amyloid core form intermolecular contacts in a 'Head-to-Head', 'Tail-to-Tail' fashion, but the 'Central Core' is sequestered through intramolecular contacts. The Head acquires productive interactions first, and these nucleate assembly. Variations in the length of the amyloid core and the nature of intermolecular interfaces form the structural basis of distinct prion 'strains', which produce variant phenotypes in vivo. These findings resolve several problems in yeast prion biology and have broad implications for other amyloids.     

Maintenance of an unfolded polypeptide by a cognate chaperone in bacterial type III secretion.

Many bacterial pathogens use a type III protein secretion system to deliver virulence effector proteins directly into the host cell cytosol, where they modulate cellular processes. A requirement for the effective translocation of several such effector proteins is the binding of specific cytosolic chaperones, which typically interact with discrete domains in the virulence factors. We report here the crystal structure at 1.9 A resolution of the chaperone-binding domain of the Salmonella effector protein SptP with its cognate chaperone SicP. The structure reveals that this domain is maintained in an extended, unfolded conformation that is wound around three successive chaperone molecules. Short segments from two different SptP molecules are juxtaposed by the chaperones, where they dimerize across a hydrophobic interface. These results imply that the chaperones associated with the type III secretion system maintain their substrates in a secretion-competent state that is capable of engaging the secretion machinery to travel through the type III apparatus in an unfolded or partially folded manner.     

Mapping the conformational wave of acetylcholine receptor channel gating

Allosteric transitions allow fast regulation of protein function in living systems. Even though the end points of such conformational changes are known for many proteins, the characteristics of the paths connecting these states remain largely unexplored. Rate-equilibrium linear free-energy relationships (LFERs) provide information about such pathways by relating changes in the free energy of the transition state to those of the ground states upon systematic perturbation of the system. Here we present an LFER analysis of the gating reaction pathway of the muscle acetylcholine receptor. We studied the closed <==> open conformational change at the single-molecule level following perturbation by series of single-site mutations, agonists and membrane voltages. This method provided a snapshot of several regions of the receptor at the transition state in terms of their approximate positions along the reaction coordinate, on a scale from 0 (closed-like) to 1 (open-like). The resulting map reveals a spatial gradient of positional values, which suggests that the conformational change proceeds in a wave-like manner, with the low-to-high affinity change at the transmitter-binding sites preceding the complete opening of the pore.     

Tapping the immunological repertoire to produce antibodies of predetermined specificity

We understand the structure of antibodies in detail, but know little about the molecular basis of the immunogenicity of proteins. Recent experiments have shown that chemically synthesized peptides representative of virtually any part of the surface of a protein can elicit antibodies reactive with the native molecule. Such peptides can serve as synthetic vaccines, and antibodies, useful in the study of changes in protein structure, can be generated. As these antibodies react with regions of the protein known in advance to the experimenter, they can be said to be of predetermined specificity.     

Proline isomerism in staphylococcal nuclease characterized by NMR and site-directed mutagenesis

Nuclear magnetic resonance (NMR) studies have shown that two distinct folded conformations of staphylococcal nuclease coexist in solution and that these two states can interconvert directly without passing through an unfolded state. These experiments have also revealed that the two forms have very different folding kinetics, although the possibility that one component is an obligatory intermediate for the folding of the other form could be discounted. Here we report NMR data which show that alternative unfolded states are also distinguishable. These observations led us to hypothesize that cis/trans isomerism at a single peptide bond between a proline and its preceding residue might be the origin of the conformational multiplicity. Proline 117 was identified as a likely candidate for the site concerned and a mutant protein, in which Pro 117 was replaced by Gly, was constructed in order to test this. Alternative conformations are not observed in the spectrum of this mutant, lending powerful support to this hypothesis.