共查询到20条相似文献,搜索用时 31 毫秒
1.
Korzhnev DM Religa TL Lundström P Fersht AR Kay LE 《Journal of molecular biology》2007,372(2):497-512
The FF domain from the human protein HYPA/FBP11 folds via a low-energy on-pathway intermediate (I). Elucidation of the structure of such folding intermediates and denatured states under conditions that favour folding are difficult tasks. Here, we investigated the millisecond time-scale equilibrium folding transition of the 71-residue four-helix bundle wild-type protein by (15)N, (13)C(alpha) and methyl(13)C Carr-Purcell-Meiboom-Gill (CPMG) NMR relaxation dispersion experiments and by (1)H/(2)H-exchange measurements. The relaxation data for the wild-type protein fitted a simple two-site exchange process between the folded state (F) and I. Destabilization of F in mutants A17G and Q19G allowed the detection of the unfolded state U by (15)N CPMG relaxation dispersion. The dispersion data for these mutants fitted a three-site exchange scheme, U<-->I<-->F, with I populated higher than U. The kinetics and thermodynamics of the folding reaction were obtained via temperature and urea-dependent relaxation dispersion experiments, along with structural information on I from backbone (15)N, (13)C(alpha) and side-chain methyl (13)C chemical shifts, with further information from protection factors for the backbone amide groups from (1)H/(2)H-exchange. Notably, helices H1-H3 are at least partially formed in I, while helix H4 is largely disordered. Chemical shift differences for the methyl (13)C nuclei suggest a paucity of stable, native-like hydrophobic interactions in I. These data are consistent with Phi-analysis of the rate-limiting transition state between I and F. The combination of relaxation dispersion and Phi data can elucidate whole experimental folding pathways. 相似文献
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Wong KB Clarke J Bond CJ Neira JL Freund SM Fersht AR Daggett V 《Journal of molecular biology》2000,296(5):1257-1282
The detailed characterization of denatured proteins remains elusive due to their mobility and conformational heterogeneity. NMR studies are beginning to provide clues regarding residual structure in the denatured state but the resulting data are too sparse to be transformed into molecular models using conventional techniques. Molecular dynamics simulations can complement NMR by providing detailed structural information for components of the denatured ensemble. Here, we describe three independent 4 ns high-temperature molecular dynamics simulations of barnase in water. The simulated denatured state was conformationally heterogeneous with respect to the conformations populated both within a single simulation and between simulations. Nonetheless, there were some persistent interactions that occurred to varying degrees in all simulations and primarily involved the formation of fluid hydrophobic clusters with participating residues changing over time. The region of the beta(3-4) hairpin contained a particularly high degree of such side-chain interactions but it lacked beta-structure in two of the three denatured ensembles: beta(3-4) was the only portion of the beta-structure to contain significant residual structure in the denatured state. The two principal alpha-helices (alpha1 and alpha2) adopted dynamic helical structure. In addition, there were persistent contacts that pinched off core 2 from the body of the protein. The rest of the protein was unstructured, aside from transient and mostly local side-chain interactions. Overall, the simulated denatured state contains residual structure in the form of dynamic, fluctuating secondary structure in alpha1 and alpha2, as well as fluctuating tertiary contacts in the beta(3-4) region, and between alpha1 and beta(3-4), in agreement with previous NMR studies. Here, we also show that these regions containing residual structure display impaired mobility by both molecular dynamics and NMR relaxation experiments. The residual structure was important in decreasing the conformational states available to the chain and in repairing disrupted regions. For example, tertiary contacts between beta(3-4) and alpha1 assisted in the refolding of alpha1. This contact-assisted helix formation was confirmed in fragment simulations of beta(3-4) and alpha1 alone and complexed, and, as such, alpha1 and beta(3-4) appear to be folding initiation sites. The role of these sites in folding was investigated by working backwards and considering the simulation in reverse, noting that earlier time-points from the simulations provide models of the major intermediate and transition states in quantitative agreement with data from both unfolding and refolding experiments. Both beta(3-4) and alpha1 are dynamic in the denatured state but when they collide and make enough contacts, they provide a loose structural scaffold onto which further beta-strands pack. The beta-structure condenses about beta(3-4), while alpha1 aids in stabilizing beta(3-4) and maintaining its orientation. The resulting beta-structure is relatively planar and loose in the major intermediate. Further packing ensues, and as a result the beta-sheet twists, leading to the major transition state. The structure is still expanded and loops are not well formed at this point. Fine-tuning of the packing interactions and the final condensation of the structure then occurs to yield the native state. 相似文献
5.
Tollinger M Kloiber K Agoston B Dorigoni C Lichtenecker R Schmid W Konrat R 《Biochemistry》2006,45(29):8885-8893
NMR relaxation dispersion techniques were used to investigate conformational exchange of the three-helix bundle protein KIX under native conditions. These experiments provide site-resolved kinetic information about microsecond-to-millisecond time scale motions along with structural (chemical shift) information without requiring a perturbation of the equilibrium. All kinetic data are consistent with an apparent two-state transition between natively folded KIX and a partially unfolded high-energy state that is populated to 3.0 +/- 0.2% at 27 degrees C. By combining (13)C- and (15)N-based experiments that probe specific structural aspects, we show that the sparsely populated high-energy state displays a strong conformational preference. An isolated secondary structural element, C-terminal helix alpha3, is highly populated, while the hydrophobic core of the domain and the remainder of the protein backbone, including helices alpha1 and alpha2, are disordered and devoid of specific interactions. This high-energy state presumably represents the equilibrium analogue of a folding intermediate that is transiently populated in stopped-flow kinetic experiments [Horng, J. C., Tracz, S. M., Lumb, K. J., and Raleigh, D. P. (2002) Biochemistry 44, 627-634]. 相似文献
6.
Structures of intermediates and transition states in protein folding are usually characterized by amide hydrogen exchange and protein engineering methods and interpreted on the basis of the assumption that they have native-like conformations. We were able to stabilize and determine the high-resolution structure of a partially unfolded intermediate that exists after the rate-limiting step of a four-helix bundle protein, Rd-apocyt b(562), by multidimensional NMR methods. The intermediate has partial native-like secondary structure and backbone topology, consistent with our earlier native state hydrogen exchange results. However, non-native hydrophobic interactions exist throughout the structure. These and other results in the literature suggest that non-native hydrophobic interactions may occur generally in partially folded states. This can alter the interpretation of mutational protein engineering results in terms of native-like side chain interactions. In addition, since the intermediate exists after the rate-limiting step and Rd-apocyt b(562) folds very rapidly (k(f) approximately 10(4) s(-1)), these results suggest that non-native hydrophobic interactions, in the absence of topological misfolding, are repaired too rapidly to slow folding and cause the accumulation of folding intermediates. More generally, these results illustrate an approach for determining the high-resolution structure of folding intermediates. 相似文献
7.
Kunihiro Kuwajima 《Journal of molecular biology》1977,114(2):241-258
It has been shown that α-lactalbumin undergoes a three-state denaturation, involving a helical intermediate state, on treatment with guanidine hydrochloride. The unfolding of the protein and the characteristics of the intermediate state are examined by means of circular dichroism, difference spectra and pH-jump measurements to investigate the temperature dependence and kinetic properties of the unfolding and refolding, the pH dependence of the transition between the intermediate and the fully unfolded states, and the effect of disulphide bond reduction on the stabilization of the intermediate.The results show that the long-range specific interactions such as specific electrostatic interactions and disulphide linkages are not important for stabilizing the intermediate, and that the transition between the intermediate and the fully unfolded states is extremely rapid (a relaxation time of less than one millisecond) and may correspond to the helix-coil transition of a polypeptide backbone. On the other hand, the activation parameters of the transition between the native and the intermediate states have suggested that the final stabilization by charge-pair interactions is preceded by hydrophobic interactions in the process of going from the intermediate to the native state.The mechanism of folding of the protein is discussed, and the folding process from the fully unfolded to the native state is apparently divided into at least three main steps: (1) the formation of incipient helical structures dictated by local interactions; (2) the packing of the helical segments accompanied with hydrophobic interactions; (3) the final stabilization by the electrostatic interactions. The relevance to the current theoretical results on protein folding is also discussed. 相似文献
8.
Ahn HC Le YT Nagchowdhuri PS Derose EF Putnam-Evans C London RE Markley JL Lim KH 《Protein science : a publication of the Protein Society》2006,15(11):2552-2557
Amyloid formation is associated with structural changes of native polypeptides to monomeric intermediate states and their self-assembly into insoluble aggregates. Characterizations of the amyloidogenic intermediate state are, therefore, of great importance in understanding the early stage of amyloidogenesis. Here, we present NMR investigations of the structural and dynamic properties of the acid-unfolded amyloidogenic intermediate state of the phosphatidylinositol 3-kinase (PI3K) SH3 domain--a model peptide. The monomeric amyloidogenic state of the SH3 domain studied at pH 2.0 (35 degrees C) was shown to be substantially disordered with no secondary structural preferences. (15)N NMR relaxation experiments indicated that the unfolded polypeptide is highly flexible on a subnanosecond timescale when observed under the amyloidogenic condition (pH 2.0, 35 degrees C). However, more restricted motions were detected in residues located primarily in the beta-strands as well as in a loop in the native fold. In addition, nonnative long-range interactions were observed between the residues with the reduced flexibility by paramagnetic relaxation enhancement (PRE) experiments. These indicate that the acid-unfolded state of the SH3 domain adopts a partly folded conformation through nonnative long-range contacts between the dynamically restricted residues at the amyloid-forming condition. 相似文献
9.
Neudecker P Zarrine-Afsar A Choy WY Muhandiram DR Davidson AR Kay LE 《Journal of molecular biology》2006,363(5):958-976
Recent 15N and 13C spin-relaxation dispersion studies of fast-folding mutants of the Fyn SH3 domain have established that folding proceeds through a low-populated on-pathway intermediate (I) where the central beta-sheet is at least partially formed, but without interactions between the NH2- and COOH-terminal beta-strands that exist in the folded state (F). Initial studies focused on mutants where Gly48 is replaced; in an effort to establish whether this intermediate is a general feature of Fyn SH3 folding a series of 15N relaxation experiments monitoring the folding of Fyn SH3 mutants N53P/V55L and A39V/N53P/V55L are reported here. For these mutants as well, folding proceeds through an on-pathway intermediate with similar features to those observed for G48M and G48V Fyn SH3 domains. However, the 15N chemical shifts extracted for the intermediate indicate pronounced non-native contacts between the NH2 and COOH-terminal regions not observed previously. The kinetic parameters extracted for the folding of A39V/N53P/V55L Fyn SH3 from the three-state folding model F<-->I<-->U are in good agreement with folding and unfolding rates extrapolated to zero denaturant obtained from stopped-flow experiments analyzed in terms of a simplified two-state folding reaction. The folding of the triple mutant was studied over a wide range of temperatures, establishing that there is no difference in heat capacities between F and I states. This confirms a compact folding intermediate structure, which is supported by the 15N chemical shifts of the I state extracted from the dispersion data. The temperature-dependent relaxation data simplifies data analysis because at low temperatures (< 25 degrees C) the unfolded state (U) is negligibly populated relative to I and F. A comparison between parameters extracted at low temperatures where the F<-->I exchange model is appropriate with those from the more complex, three-state model at higher temperatures has been used to validate the protocol for analysis of three-site exchange relaxation data. 相似文献
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We report high temperature molecular dynamics simulations of the unfolding of the TRPZ1 peptide using an explicit model for the solvent. The system has been simulated for a total of 6 μs with 100-ns minimal continuous stretches of trajectory. The populated states along the simulations are identified by monitoring multiple observables, probing both the structure and the flexibility of the conformations. Several unfolding and refolding transition pathways are sampled and analyzed. The unfolding process of the peptide occurs in two steps because of the accumulation of a metastable on-pathway intermediate state stabilized by two native backbone hydrogen bonds assisted by nonnative hydrophobic interactions between the tryptophan side chains. Analysis of the un/folding kinetics and classical commitment probability calculations on the conformations extracted from the transition pathways show that the rate-limiting step for unfolding is the disruption of the ordered native hydrophobic packing (Trp-zip motif) leading from the native to the intermediate state. But, the speed of the folding process is mainly determined by the transition from the completely unfolded state to the intermediate and specifically by the closure of the hairpin loop driven by formation of two native backbone hydrogen bonds and hydrophobic contacts between tryptophan residues. The temperature dependence of the unfolding time provides an estimate of the unfolding activation enthalpy that is in agreement with experiments. The unfolding time extrapolated to room temperature is in agreement with the experimental data as well, thus providing a further validation to the analysis reported here. 相似文献
12.
Based on the C(alpha) Go-type model, the folding kinetics and mechanisms of protein ubiquitin with mixed alpha/beta topology are studied by molecular dynamics simulations. The relaxation kinetics shows that there are three phases, namely the major phase, the intermediate phase and the slowest minor phase. The existence of these three phases are relevant to the phenomenon found in experiments. According to our simulations, the folding at high temperatures around the folding transition temperature T(f) is of a two-state process, and the folding nucleus is consisted of contacts between the front end of alpha-helix and the turn(4). The folding at low temperature (approximately T = 0.8) is also studied, where an A-state like structure is found lying on the major folding pathway. The appearance of this structure is related to the stability of the first part (residue 1-51) of protein ubiquitin. As the temperature decreases, the formation of secondary structures, tertiary structures and collapse of the protein are found to be decoupled gradually and the folding mechanism changes from the nucleation-condensation to the diffusion-collision. This feature indicates a unifying common folding mechanism for proteins. The intermediate phase is also studied and is found to represent a folding process via a long-lived intermediate state which is stabilized by strong interactions between the beta(1) and the beta(5) strand. These strong interactions are important for the function of protein ubiquitin as a molecular chaperone. Thus the intermediate phase is assumed as a byproduct of the requirement of protein function. In addition, the validity of the current Go-model is also investigated, and a lower limited temperature for protein ubiquitin T(limit) = 0.8 is proposed. At temperatures higher than this value, the kinetic traps due to glass dynamics cannot be significantly populated and the intermediate states can be reliably identified although there is slight chevron rollover in the folding rates. At temperature lower than T(limit), however, the traps due to glass dynamics become dominant and may be mistaken for real intermediate states. This limitation of valid temperature range prevents us to reveal the burst phase intermediate in the major folding phase since it might only be stabilized at temperatures lower than T(limit), according to experiments. Our works show that caution must be taken when studying low-temperature intermediate states by using the C(alpha) Go-models. 相似文献
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Nichole E. O’Connell Michael J. Grey Yuefeng Tang Pallav Kosuri Vesselin Z. Miloushev Daniel P. Raleigh Arthur G. Palmer III 《Journal of biomolecular NMR》2009,45(1-2):85-98
Identification and characterization of ensembles of intermediate states remains an important objective in describing protein folding in atomic detail. The 67-residue villin headpiece, HP67, consists of an N-terminal subdomain (residues 10–42) that transiently unfolds at equilibrium under native-like conditions and a highly stable C-terminal subdomain (residues 43–76). The transition between folded and unfolded states of the N-terminal domain has been characterized previously by 15N NMR relaxation dispersion measurements (Grey et al. in J Mol Biol 355:1078, 2006). In the present work, 13C spin relaxation was used to further characterize backbone and hydrophobic core contributions to the unfolding process. Relaxation of 13Cα spins was measured using the Hahn echo technique at five static magnetic fields (11.7, 14.1, 16.4, 18.8, and 21.1 T) and the Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion method at a static magnetic field of 14.1 T. Relaxation of methyl 13C spins was measured using CPMG relaxation dispersion experiments at static magnetic fields of 14.1 and 18.8 T. Results for 13C and 15N spins yielded a consistent model in which the partially unfolded intermediate state of the N-terminal subdomain maintains residual structure for residues near the unprotonated His41 imidazole ring and in the interface between the N- and C-terminal subdomains. In addition, a second faster process was detected that appears to represent local dynamics within the folded state of the molecule and is largely confined to the hydrophobic interface between the N- and C-terminal subdomains. 相似文献
15.
Hydration and packing along the folding pathway of SH3 domains by pressure-dependent NMR 总被引:2,自引:0,他引:2
The volumetric properties associated with protein folding transitions reflect changes in protein packing and hydration of the states that participate in the folding reaction. Here, NMR spin relaxation techniques are employed to probe the folding-unfolding kinetics of two SH3 domains as a function of pressure so that the changes in partial molar volumes along the folding pathway can be measured. The two domains fold with rates that differ by approximately 3 orders of magnitude, so their folding dynamics must be probed using different NMR relaxation experiments. In the case of the drkN SH3 domain that folds via a two-state mechanism on a time scale of seconds, nitrogen magnetization exchange spectroscopy is employed, while for the G48M mutant of the Fyn SH3 domain where the folding occurs on the millisecond time scale (three-step reaction), relaxation dispersion experiments are utilized. The NMR methodology is extremely sensitive to even small changes in equilibrium and rate constants, so reliable estimates of partial molar volumes can be obtained using low pressures (1-120 bar), thus minimizing perturbations to any of the states along the folding reaction coordinate. The volumetric data that were obtained are consistent with a similar folding mechanism for both SH3 domains, involving early chain compaction to states that are at least partially hydrated. This work emphasizes the role of NMR spin relaxation in studying dynamic processes over a wide range of time scales. 相似文献
16.
Site-directed mutagenesis has been used to probe the interactions that stabilize the equilibrium and burst phase kinetic intermediates formed by apomyoglobin. Nine bulky hydrophobic residues in the A, E, G and H helices were replaced by alanine, and the effects on protein stability and kinetic folding pathways were determined. Hydrogen exchange pulse-labeling experiments, with NMR detection, were performed for all mutants. All of the alanine substitutions resulted in changes in proton occupancy or an increased rate of hydrogen-deuterium exchange for amides in the immediate vicinity of the mutation. In addition, most mutations affected residues in distant parts of the amino acid sequence, providing insights into the topology of the burst phase intermediate and the interactions that stabilize its structure. Differences between the pH 4 equilibrium molten globule and the kinetic intermediate are evident: the E helix region plays no discernible role in the equilibrium intermediate, but contributes significantly to stabilization of the ensemble of compact intermediates formed during kinetic refolding. Mutations that interfere with docking of the E helix onto the preformed A/B/G/H helix core substantially decrease the folding rate, indicating that docking and folding of the E helix region occurs prior to formation of the apomyoglobin folding transition state. The results of the mutagenesis experiments are consistent with rapid formation of an ensemble of compact burst phase intermediates with an overall native-like topological arrangement of the A, B, E, G, and H helices. However, the experiments also point to disorder in docking of the E helix and to non-native contacts in the kinetic intermediate. In particular, there is evidence for translocation of the H helix by approximately one helical turn towards its N terminus to maximize hydrophobic interactions with helix G. Thus, the burst phase intermediate observed during kinetic refolding of apomyoglobin consists of an ensemble of compact, kinetically trapped states in which the helix docking appears to be topologically correct, but in which there are local non-native interactions that must be resolved before the protein can fold to the native structure. 相似文献
17.
Collapse of unfolded protein chains is an early event in folding. It affects structural properties of intrinsically disordered proteins, which take a considerable fraction of the human proteome. Collapse is generally believed to be driven by hydrophobic forces imposed by the presence of nonpolar amino acid side chains. Contributions from backbone hydrogen bonds to protein folding and stability, however, are controversial. To date, the experimental dissection of side-chain and backbone contributions has not yet been achieved because both types of interactions are integral parts of protein structure. Here, we realized this goal by applying mutagenesis and chemical modification on a set of disordered peptides and proteins. We measured the protein dimensions and kinetics of intra-chain diffusion of modified polypeptides at the level of individual molecules using fluorescence correlation spectroscopy, thereby avoiding artifacts commonly caused by aggregation of unfolded protein material in bulk. We found no contributions from side chains to collapse but, instead, identified backbone interactions as a source sufficient to form globules of native-like dimensions. The presence of backbone hydrogen bonds decreased polypeptide water solubility dramatically and accelerated the nanosecond kinetics of loop closure, in agreement with recent predictions from computer simulation. The presence of side chains, instead, slowed loop closure and modulated the dimensions of intrinsically disordered domains. It appeared that the transient formation of backbone interactions facilitates the diffusive search for productive conformations at the early stage of folding and within intrinsically disordered proteins. 相似文献
18.
Src homology 3 (SH3) domains are small modules that are thought to fold via a two-state mechanism, without the accumulation of significant populations of intermediate states. Relaxation dispersion NMR studies of the folding of G48V and G48M mutants of the Fyn SH3 domain have established that, at least for these modules, folding proceeds through the formation of a transient on-pathway intermediate with an equilibrium population of 1-2% that can be readily detected [Korzhnev, D. M., et al. (2004) Nature 430, 586-590]. To investigate the generality of this result, we present an (15)N relaxation dispersion NMR study of a pair of additional SH3 domains, including a G48V mutant of a stabilized Abp1p SH3 domain that shares 36% sequence identity with the Fyn SH3 module, and a A39V/N53P/V55L mutant Fyn SH3 domain. A transient folding intermediate is detected for both of the proteins studied here, and the dispersion data are well fit to a folding model of the form F <--> I <--> U, where F, I, and U correspond to folded, intermediate, and unfolded states, respectively. The temperature dependencies of the folding/unfolding rate constants were obtained so that the thermodynamic properties of each of F, I, and U could be established. The detection of I states in folding pathways of all SH3 domains examined to date via relaxation dispersion NMR spectroscopy indicates that such intermediates may well be a conserved feature in the folding of such domains in general but that their transient nature along with their low population makes detection difficult using more well-established approaches to the study of folding. 相似文献
19.
Travaglini-Allocatelli C Gianni S Morea V Tramontano A Soulimane T Brunori M 《The Journal of biological chemistry》2003,278(42):41136-41140
Understanding the role of partially folded intermediate states in the folding mechanism of a protein is a crucial yet very difficult problem. We exploited a kinetic approach to demonstrate that a transient intermediate of a thermostable member of the widely studied cytochrome c family (cytochrome c552 from Thermus thermophilus) is indeed on-pathway. This is the first clear indication of an obligatory intermediate in the folding mechanism of a cytochrome c. The fluorescence properties of this intermediate demonstrate that the relative position of the heme and of the only tryptophan residue cannot correspond to their native orientation. Based on an analysis of the three-dimensional structure of cytochrome c552, we propose an interpretation of the data which explains the residual fluorescence of the intermediate and is consistent with the established role played by some conserved interhelical interactions in the folding of other members of this family. A limited set of topologically conserved contacts may guide the folding of evolutionary distant cytochromes c through the same partially structured state, which, however, can play different kinetic roles, acting either as an intermediate or a transition state. 相似文献
20.
An accurate characterization of the transition state ensemble (TSE) is central to furthering our understanding of the protein folding reaction. We have extensively tested a recently reported method for studying a protein's TSE, utilizing phi-value data from protein engineering experiments and computational studies as restraints in all-atom Monte Carlo (MC) simulations. The validity of interpreting experimental phi-values as the fraction of native contacts made by a residue in the TSE was explored, revealing that this definition is unable to uniquely specify a TSE. The identification of protein G's second hairpin, in both pre and post-transition conformations demonstrates that high experimental phi-values do not guarantee a residue's importance in the TSE. An analysis of simulations based on structures restrained by experimental phi-values is necessary to yield this result, which is not obvious from a simplistic interpretation of individual phi-values. The TSE that we obtain corresponds to a single, specific nucleation event, characterized by six residues common to all three observed, convergent folding pathways. The same specific nucleus was independently identified from computational and experimental data, and "Conservation of Conservation" analysis in the protein G fold. When associated strictly with complete nucleus formation and concomitant chain collapse, folding is a well-defined two state event. Once the nucleus has formed, the folding reaction enters a slow relaxation process associated with side-chain packing and small, local backbone rearrangements. A detailed analysis of phi-values and their relationship to the transition state ensemble allows us to construct a unified theoretical model of protein G folding. 相似文献