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1.
To test the hypothesis that the folding pathways of evolutionarily related proteins with similar three-dimensional structures but widely different sequences should be similar, the folding pathway of apoleghemoglobin has been characterized using stopped-flow circular dichroism, heteronuclear NMR pulse labeling techniques and mass spectrometry. The pathway of folding was found to differ significantly from that of a protein of the same family, apomyoglobin, although both proteins appear to fold through helical burst phase intermediates. For leghemoglobin, the burst phase intermediate exhibits stable helical structure in the G and H helices, together with a small region in the center of the E helix. The A and B helices are not stabilized until later stages of the folding process. The structure of the burst phase folding intermediate thus differs from that of apomyoglobin, in which stable helical structure is formed in the A, B, G and H helix regions.  相似文献   

2.
Measurements of the stability as a function of pH for the acyl-coenzyme A binding protein (ACBP) has shown a significant difference in the pH transition midpoint measured by NMR spectroscopy at pH 3.12 and the transition midpoint measured at pH 2.92 and 2.97 by circular dichroism and by fluorescence spectroscopy, respectively. A similar behavior has not been observed in other proteins. It is suggested that these differences arise because the population of the unfolded molecules still contains significant amounts of native like secondary and tertiary structure. NMR spectroscopy measures the concentration of the two components of the folding unfolding equilibrium individually, whereas circular dichroism and fluorescence measure the concentration of the conformations of the light-absorbing chromophores present in both the folded and the unfolded molecules. In the narrow pH range, nascent structure can be detected as the average amount of secondary structure per unfolded molecule and hydrophobic interactions in the population of unfolded molecules. These structures are not observable immediately by NMR spectroscopy; however, a chemical shift analysis of the peptide backbone (13)C chemical shift indicates strongly the existence of short-lived and transient helical structures at pH 2.3. Magnetization transfer studies have been applied to study the equilibrium between folded and unfolded ACBP near the pH transition point measured by NMR. This study has shown that there are two categories of subpopulations in the population of unfolded ACBP. One for which magnetization can be transferred to the folded form during the folding process, and one for which transfer is not observed. The molecules of the latter population of unfolded protein apparently, do not fold within the time-frame of the magnetization transfer experiment. This result suggests the existence of a subpopulation of the acid-unfolded protein molecules with a high propensity for folding. It is suggested that in this subpopulation, a particular set of native like interactions in the peptide backbone and between side-chains in the peptide chain have to be formed.  相似文献   

3.
Paramagnetic relaxation enhancement measurements in the denatured state of ACBP have provided distance restraints that have been used in computer simulations to determine the conformational ensembles representing the denatured states of ACBP under a variety of conditions. A detailed comparison of the residual structure in the denatured state of ACBP under these different conditions has enabled us to infer that regions in the N and C-terminal parts of the protein sequence have a high tendency to interact in the unfolded state under physiological conditions. By comparing the structural features in the denatured states with those in the transition state for folding we also provided new insights into the mechanism of formation of the native state of this protein.  相似文献   

4.
The folding pathways of four mutants in which bulky hydrophobic residues in the B helix of apomyoglobin (ApoMb) are replaced by alanine (I28A, L29A, I30A, and L32A) have been analyzed using equilibrium and kinetic methods employing NMR, CD, fluorescence and mass spectrometry. Hydrogen exchange pulse-labeling followed by mass spectrometry reveals detectable intermediates in the kinetic folding pathways of each of these mutants. Comparison of the quench-flow data analyzed by NMR for the wild-type protein and the mutants showed that the substitutions I28A, L29A and L32A lead to destabilization of the B helix in the burst phase kinetic intermediate, relative to wild-type apomyoglobin. In contrast, the I30A mutation apparently has a slight stabilizing effect on the B helix in the burst phase intermediate; under weak labeling conditions, residues in the C helix region were also relatively stabilized in the mutant compared to the wild-type protein. This suggests that native-like helix B/helix C packing interactions occur in the folding intermediate. The L32A mutant showed significantly lower proton occupancies in the burst phase for several residues in the G helix, specifically F106, I107, E109 and A110, which are in close proximity to L32 in the X-ray structure of myoglobin, providing direct evidence that native-like helix B/helix G contacts are formed in the apomyoglobin burst phase intermediate. The L29A mutation resulted in an increase in burst phase proton occupancies for several residues in the E helix. Since these regions of the B and E helices are not in contact in the native myoglobin structure, these effects suggest the possibility of non-native B/E packing interactions in the kinetic intermediate. The differing effects of these B helix mutations on the apomyoglobin folding process suggests that each side-chain plays a different and important role in forming stable structure in the burst phase intermediate, and points to a role for both native-like and non-native contacts in stabilization of the folding intermediate.  相似文献   

5.
6.
The globular 22-kDa protein UMP/CMP from Dictyostelium discoideum (UmpK) belongs to the family of nucleoside monophosphate (NMP) kinases. These enzymes not only show high sequence and structure similarities but also share the α/β-fold, a very common protein topology. We investigated the protein folding mechanism of UmpK as a representative for this ubiquitous enzyme class. Equilibrium stability towards urea and the unfolding and refolding kinetics were studied by means of fluorescence and far-UV CD spectroscopy. Although the unfolding can be described by a two-state process, folding kinetics are rather complex with four refolding phases that can be resolved and an additional burst phase. Moreover, two of these phases exhibit a pronounced rollover in the refolding limb that cannot be explained by aggregation. Whilst secondary structure formation is not observed in the burst phase reaction, folding to the native structure is strongly influenced by the slowest phase, since 30% of the α-helical CD signal is restored therein. This process can be assigned to proline isomerization and is strongly accelerated by the Escherichia coli peptidyl-prolyl isomerase trigger factor. The analysis of our single-mixing and double-mixing experiments suggests the occurrence of an off-pathway intermediate and an unproductive collapsed structure, which appear to be rate limiting for the folding of UmpK.  相似文献   

7.
Residual dipolar couplings in the denatured state of bovine acyl-coenzyme A binding protein (ACBP) oriented in strained polyacrylamide gels have been shown to be a sensitive, sequence-specific probe for residual secondary structure. Results supporting this were obtained by comparing residual dipolar couplings under different denaturing conditions. The data were analyzed using the program molecular fragment replacement (MFR), which demonstrated alpha-helix propensity in four isolated stretches along the protein backbone, and these coincide with the location of native helices. This is in full agreement with earlier findings based on secondary chemical shift values. Furthermore, N-H residual dipolar couplings provided direct evidence for the existence of native-like hydrophobic interactions in the acid-denatured state of ACBP at pH 2.3. It was shown that replacement of the hydrophobic side-chain of residue Ile27 with alanine in helix A2 leads to large decreases of residual dipolar couplings in residues that form helix A4 in the native state. It is suggested that the Ile to Ala mutation changes the probability for the formation of long-range interactions, which are present in the acid-denatured state of the wild-type protein. These long-range interactions are similar to those proposed to form in the transition state of folding of ACBP. Therefore, the application of residual dipolar couplings in combination with a comparative mutation study has demonstrated the presence of precursors to the folding transition state under acid-unfolding conditions.  相似文献   

8.
R67 dihydrofolate reductase (DHFR) is a homotetrameric enzyme. Its subunit has a core structure consisting of five antiparallel beta-strands that form a compact beta-barrel. Our interest was to describe the molecular mechanism of the complete folding pathway of this beta-sheet protein, focusing on how the oligomerization steps are coordinated with the formation of secondary and tertiary structures all along the folding process. The folding kinetics of R67 dihydrofolate reductase into dimers at pH 5.0 were first examined by intrinsic tryptophan fluorescence, fluorescence energy transfer, and circular dichroism spectroscopy. The process was shown to consist of at least four steps, including a burst, a rapid, a medium, and a slow phase. Measurements of the ellipticity at 222 nm indicated that about 50% of the total change associated with refolding occurred during the 4 ms dead time of the stopped-flow instrument, indicating a substantial burst of secondary structure. The bimolecular association step was detected using fluorescence energy transfer and corresponded to the rapid phase. The slow phase was attributed to a rate-limiting isomerization of peptidyl-prolyl bonds involving 15% of the unfolded population. A complete folding pathway from the unfolded monomer to the native tetramer was proposed and an original model based upon the existence of early partially folded monomeric intermediates, rapidly stabilized in a dimeric form able to self-associate into the native homotetramer was formulated. The rate constants of these various steps were determined by fitting the kinetic traces to this model and supported our mechanistic assumptions.  相似文献   

9.
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   

10.
The time-course of monovalent cation-induced folding of the L-21 Sca1 Tetrahymena thermophila ribozyme and a selected mutant was quantitatively followed using synchrotron X-ray (.OH) footprinting. Initiating folding by increasing the concentration of either Na+ or K+ to 1.5M from an initial condition of approximately 0.008 M Na+ at 42 degrees C resulted in the complete formation of tertiary contacts within the P5abc subdomain and between the peripheral helices within the dead time of our measurements (k>50 s(-1)). These results contrast with folding rates of 2-0.2 s(-1) previously observed for formation of these contacts in 10mM Mg2+ from the same initial condition. Thus, the initial formation of native tertiary contacts is inhibited by divalent but not monovalent cations. The native contacts within the catalytic core form without a detectable burst phase at rates of 0.4-1.0 s(-1) in a manner reminiscent of the Mg2+-dependent folding behavior, although tenfold faster. The tertiary interactions stabilizing the catalytic core interaction with P4-P6 and P2.1, as well as one of the protections internal for the P4-P6 domain, display progress curves with appreciable burst amplitudes and a phase comparable in rate to that of the catalytic core. That the slow folding of the ribozyme's core is a consequence of the alt-P3 secondary structure is shown by the 100% burst phase amplitudes that are observed for folding of the U273A mutant ribozyme within which the native secondary structure (P3) is strengthened. Thus, formation of a misfolded intermediate(s) resulting from the alt-P3 secondary structure is independent of ion valency while the rate at which the respective intermediates are resolved is sensitive to ion valency. The overall portrait painted by these results is that ion valency differentially affects steps in the folding process and that folding in monovalent ion alone for the U273A mutant Tetrahymena ribozyme is fast and direct.  相似文献   

11.
An important question in protein folding is whether molten globule states formed under equilibrium conditions are good structural models for kinetic folding intermediates. The structures of the kinetic and equilibrium intermediates in the folding of the plant globin apoleghemoglobin have been compared at high resolution by quench-flow pH-pulse labeling and interrupted hydrogen/deuterium exchange analyzed in dimethyl sulfoxide. Unlike its well studied homolog apomyoglobin, where the equilibrium and kinetic intermediates are quite similar, there are striking structural differences between the intermediates formed by apoleghemoglobin. In the kinetic intermediate, formed during the burst phase of the quench-flow experiment, protected amides and helical structure are found mainly in the regions corresponding to the G and H helices of the folded protein, and in parts of the E helix and CE loop regions, whereas in the equilibrium intermediate, amide protection and helical structure are seen in parts of the A and B helix regions, as well as in the G and H regions, and the E helix remains largely unfolded. These results suggest that the structure of the molten globule intermediate of apoleghemoglobin is more plastic than that of apomyoglobin, so that it is readily transformed depending on the solution conditions, particularly pH. Thus, in the case of apoleghemoglobin at least, the equilibrium molten globule formed under destabilizing conditions at acid pH is not a good model for the compact intermediate formed during kinetic refolding experiments. Our high-precision kinetic analysis also reveals an additional slow phase during the folding of apoleghemoglobin, which is not observed for apomyoglobin. Hydrogen exchange pulse-labeling experiments show that the slow-folding phase is associated with residues in the CE loop, which probably forms non-native structure in the intermediate that must be resolved before folding can proceed to completion.  相似文献   

12.
Factors governing the folding pathways and the stability of apomyoglobin have been examined by replacing the distal histidine at position 64 with phenylalanine (H64F). Acid and urea-induced unfolding experiments using CD and fluorescence techniques reveal that the mutant H64F apoprotein is significantly more stable than wild-type apoMb. Kinetic refolding studies of this variant also show a significant difference from wild-type apoMb. The amplitude of the burst phase ellipticity in stopped-flow CD measurements is increased over that of wild-type, an indication that the secondary structure content of the earliest kinetic intermediate is greater in the mutant than in the wild-type protein. In addition, the overall rate of folding is markedly increased. Hydrogen exchange pulse labeling was used to establish the structure of the initial intermediate formed during the burst phase of the H64F mutant. NMR analysis of the samples obtained at different refolding times indicates that the burst phase intermediate contains a stabilized E helix as well as the A, G, and H helices previously found in the wild-type kinetic intermediate. Replacement of the polar distal histidine residue with a nonpolar residue of similar size and shape appears to stabilize the E helix in the early stages of folding due to improved hydrophobic packing. The presence of a hydrophilic histidine at position 64 thus exacts a price in the stability and folding efficiency of the apoprotein, but this residue is nevertheless highly conserved among myoglobins due to its importance in function.  相似文献   

13.
The conformational stability and kinetics of refolding and unfolding of the W290F mutant of TEM-1 beta-lactamase have been determined as a function of guanidinium chloride concentration. The activity and spectroscopic properties of the mutant enzyme did not differ significantly from those of the wild type, indicating that the mutation has only a very limited effect on the structure of the protein. The stability of the folded protein is reduced, however, by 5-10 kJ mol-1 relative to that of the molten globule intermediate (H), but the values of the folding rate constants are unchanged, suggesting that Trp-290 becomes organized in its nativelike environment only after the rate-limiting step; i.e., the C-terminal region of the enzyme folds very late. In contrast to the significant increase in fluorescence intensity seen in the dead time (3-4 ms) of refolding of the wild-type protein, no corresponding burst phase was observed with the mutant enzyme, enabling the burst phase to be attributed specifically to the C-terminal Trp-290. This residue is suggested to be buried in a nonpolar environment from which it has to escape during subsequent folding steps. With both proteins, fast early collapse leads to a folding intermediate in which the C-terminal region of the polypeptide chain is trapped in a non-native structure, consistent with a nonhierarchical folding process.  相似文献   

14.
Recombinant microbial transglutaminase has been expressed in Escherichia coli as insoluble inclusion bodies. After we searched for refolding conditions, refolding of the protein could be done by first dilution of the unfolded enzyme in a buffer at pH 4.0, and then by titration of the pH from 4.0 to 6.0. CD analysis showed that a burst of secondary structure formation occurred within the dead time of the experiment and accounted for 75% of the signal change in the far UV CD, with little tertiary structure being formed. This burst was followed by slow rearrangement of the secondary structure accompanied by formation of tertiary structure. The secondary and tertiary structures of the final sample at pH 4.0, corresponding to the folding intermediate, were different from these structures at pH 6.0. Once the native structure was obtained, acidification of the native protein to pH 4.0 did not lead to a structure like that of the folding intermediate. Sedimentation velocity analysis showed that the folding intermediate had an expanded structure and contained no other structure species including large aggregates.  相似文献   

15.
Sasahara K  Nitta K 《Proteins》2006,63(1):127-135
The equilibrium and kinetics of folding of hen egg-white lysozyme were studied by means of CD spectroscopy in the presence of varying concentrations of ethanol under acidic condition. The equilibrium transition curves of guanidine hydrochloride-induced unfolding in 13 and 26% (v/v) ethanol have shown that the unfolding significantly deviates from a two-state mechanism. The kinetics of denaturant-induced refolding and unfolding of hen egg-white lysozyme were investigated by stopped-flow CD at three ethanol concentrations: 0, 13, and 26% (v/v). Immediately after dilution of the denaturant, the refolding curves showed a biphasic time course in the far-UV region, with a burst phase with a significant secondary structure and a slower observable phase. However, when monitored by the near-UV CD, the burst phase was not observed and all refolding kinetics were monophasic. To clarify the effect of nonnative secondary structure induced by the addition of ethanol on the folding/unfolding kinetics, the kinetic m values were estimated from the chevron plots obtained for the three ethanol concentrations. The data indicated that the folding/unfolding kinetics of hen lysozyme in the presence of varying concentrations of ethanol under acidic condition is explained by a model with both on-pathway and off-pathway intermediates of protein folding.  相似文献   

16.
Understanding the interactions between membrane proteins and the lipid bilayer is key to increasing our ability to predict and tailor the folding mechanism, structure and stability of membrane proteins. Here, we have investigated the effects of changing the membrane composition and the relative concentrations of protein and lipid on the folding mechanism of the bacterial outer membrane protein PagP. The folding pathway, monitored by tryptophan fluorescence, was found to be characterized by a burst phase, representing PagP adsorption to the liposome surface, followed by a time course that reflects the folding and insertion of the protein into the membrane. In 1,2-dilauroyl-sn-glycero-3-phosphocholine (diC(12:0)PC) liposomes, the post-adsorption time course fits well to a single exponential at high lipid-to-protein ratios (LPRs), but at low LPRs, a second exponential phase with a slower folding rate constant is observed. Interrupted refolding assays demonstrated that the two exponential phases reflect the presence of parallel folding pathways. Partitioning between these pathways was found to be modulated by the elastic properties of the membrane. Folding into mixed 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine:diC(12:0)PC liposomes resulted in a decrease in PagP adsorption to the liposomes and a switch to the slower folding pathway. By contrast, inclusion of 1,2-dilauroyl-sn-glycero-3-phosphoserine into diC(12:0)PC liposomes resulted in a decrease in the folding rate of the fast pathway. The results highlight the effect of lipid composition in tailoring the folding mechanism of a membrane protein, revealing that membrane proteins have access to multiple, competing folding routes to a unique native structure.  相似文献   

17.
A mutant of a beta-barrel protein, rat intestinal fatty acid binding protein, was predicted to be more stable than the wild-type protein due to a novel hydrogen bond. Equilibrium denaturation studies indicated the opposite: the V60N mutant protein was less stable. The folding transitions followed by CD and fluorescence were reversible and two-state for both mutant and wild-type protein. However, the rates of denaturation and renaturation of V60N were faster. During unfolding, the initial rate was associated with 75-80% of the fluorescence and all of the CD amplitude change. A subsequent rate accounted for the remaining fluorescence change for both proteins; thus the intermediate state lacked secondary structure. During folding, one rate was detected by both fluorescence and CD after an initial burst phase for both wild-type and mutant. An additional slower folding rate was detected by fluorescence for the mutant protein. The structure of the V60N mutant has been obtained and is nearly identical to prior crystal structures of IFABP. Analysis of mean differences in hydrogen bond and van der Waals interactions did not readily account for the stability loss due to the mutation. However, significant average differences of the solvent accessible surface and crystallographic displacement factors suggest entropic destabilization.  相似文献   

18.
Acyl-CoA binding protein (ACBP) maintains a pool of fatty acyl-CoA molecules in the cell and plays a role in fatty acid metabolism. The biochemical properties of Plasmodium falciparum ACBP are described together with the 2.0 A resolution crystal structures of a P. falciparum ACBP-acyl-CoA complex and of bovine ACBP in two crystal forms. Overall, the bovine ACBP crystal structures are similar to the NMR structures published previously; however, the bovine and parasite ACBP structures are less similar. The parasite ACBP is shown to have a different ligand-binding pocket, leading to an acyl-CoA binding specificity different from that of bovine ACBP. Several non-conservative differences in residues that interact with the ligand were identified between the mammalian and parasite ACBPs. These, together with measured binding-specificity differences, suggest that there is a potential for the design of molecules that might selectively block the acyl-CoA binding site.  相似文献   

19.
The acyl-CoA binding protein (ACBP) is essential for the fatty acid metabolism, membrane structure, membrane fusion, and ceramide synthesis. Here high resolution crystal structures of human cytosolic liver ACBP, unliganded and liganded with a physiological ligand, myristoyl-CoA are described. The binding of the acyl-CoA molecule induces only few structural differences near the binding pocket. The crystal form of the liganded ACBP, which has two ACBP molecules in the asymmetric unit, shows that in human ACBP the same acyl-CoA binding pocket is present as previously described for the bovine and Plasmodium falciparum ACBP and the mode of binding of the 3'-phosphate-AMP moiety is conserved. Unexpectedly, in one of the acyl-CoA binding pockets the acyl moiety is bound in a reversed mode as compared with the bovine and P. falciparum structures. In this binding mode, the myristoyl-CoA molecule is fully ordered and bound across the two ACBP molecules of the crystallographic asymmetric unit: the 3'-phosphate-AMP moiety is bound in the binding pocket of one ACBP molecule and the acyl chain is bound in the pocket of the other ACBP molecule. The remaining binding pocket cavities of these two ACBP molecules are filled by other ligand fragments. This novel binding mode shows that the acyl moiety can flip out of its classical binding pocket and bind elsewhere, suggesting a mechanism for the acyl-CoA transfer between ACBP and the active site of a target enzyme. This mechanism is of possible relevance for the in vivo function of ACBP.  相似文献   

20.
Kinetic and equilibrium studies of apomyoglobin folding pathways and intermediates have provided important insights into the mechanism of protein folding. To investigate the role of intrinsic helical propensities in the apomyoglobin folding process, a mutant has been prepared in which Asn132 and Glu136 have been substituted with glycine to destabilize the H helix. The structure and dynamics of the equilibrium molten globule state formed at pH 4.1 have been examined using NMR spectroscopy. Deviations of backbone (13)C(alpha) and (13)CO chemical shifts from random coil values reveal high populations of helical structure in the A and G helix regions and in part of the B helix. However, the H helix is significantly destabilized compared to the wild-type molten globule. Heteronuclear [(1)H]-(15)N NOEs show that, although the polypeptide backbone in the H helix region is more flexible than in the wild-type protein, its motions are restricted by transient hydrophobic interactions with the molten globule core. Quench flow hydrogen exchange measurements reveal stable helical structure in the A and G helices and part of the B helix in the burst phase kinetic intermediate and confirm that the H helix is largely unstructured. Stabilization of structure in the H helix occurs during the slow folding phases, in synchrony with the C and E helices and the CD region. The kinetic and equilibrium molten globule intermediates formed by N132G/E136G are similar in structure. Although both the wild-type apomyoglobin and the mutant fold via compact helical intermediates, the structures of the intermediates and consequently the detailed folding pathways differ. Apomyoglobin is therefore capable of compensating for mutations by using alternative folding pathways within a common basic framework. Tertiary hydrophobic interactions appear to play an important role in the formation and stabilization of secondary structure in the H helix of the N132G/E136G mutant. These studies provide important insights into the interplay between secondary and tertiary structure formation in protein folding.  相似文献   

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