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1.
The dimeric yeast protein Ure2 shows prion-like behaviour in vivo and forms amyloid fibrils in vitro. A dimeric intermediate is populated transiently during refolding and is apparently stabilized at lower pH, conditions suggested to favour Ure2 fibril formation. Here we present a quantitative analysis of the effect of pH on the thermodynamic stability of Ure2 in Tris and phosphate buffers over a 100-fold protein concentration range. We find that equilibrium denaturation is best described by a three-state model via a dimeric intermediate, even under conditions where the transition appears two-state by multiple structural probes. The free energy for complete unfolding and dissociation of Ure2 is up to 50 kcal mol(-1). Of this, at least 20 kcal mol(-1) is contributed by inter-subunit interactions. Hence the native dimer and dimeric intermediate are significantly more stable than either of their monomeric counterparts. The previously observed kinetic unfolding intermediate is suggested to represent the dissociated native-like monomer. The native state is stabilized with respect to the dimeric intermediate at higher pH and in Tris buffer, without significantly affecting the dissociation equilibrium. The effects of pH, buffer, protein concentration and temperature on the kinetics of amyloid formation were quantified by monitoring thioflavin T fluorescence. The lag time decreases with increasing protein concentration and fibril formation shows pseudo-first order kinetics, consistent with a nucleated assembly mechanism. In Tris buffer the lag time is increased, suggesting that stabilization of the native state disfavours amyloid nucleation. 相似文献
2.
The yeast non-Mendelian factor [URE3] propagates by a prion-like mechanism, involving aggregation of the chromosomally encoded protein Ure2. The [URE3] phenotype is equivalent to loss of function of Ure2, a protein involved in regulation of nitrogen metabolism. The prion-like behaviour of Ure2 in vivo is dependent on the first 65 amino acid residues of its N-terminal region which contains a highly repetitive sequence rich in asparagine. This region has been termed the prion-determining domain (PrD). Removal of as little as residues 2-20 of the protein is sufficient to prevent occurrence of the [URE3] phenotype. Removal of the PrD does not affect the regulatory activity of Ure2. The C-terminal portion of the protein has homology to glutathione S -transferases, which are dimeric proteins. We have produced the Ure2 protein to high yield in Escherichia coli from a synthetic gene. The recombinant purified protein is shown to be a dimer. The stability, folding and oligomeric state of Ure2 and a series of N-terminally truncated or deleted variants were studied and compared. The stability of Ure2, DeltaGD-N, H2O, determined by chemical denaturation and monitored by fluorescence, is 12.1(+/-0.4) kcal mol-1at 25 degrees C and pH 8.4. A range of structural probes show a single, coincident unfolding transition, which is invariant over a 550-fold change in protein concentration. The stability is the same within error for Ure2 variants lacking all or part of the prion-determining domain. The data indicate that in the folded protein the PrD is in an unstructured conformation and does not form specific intra- or intermolecular interactions at micromolar protein concentrations. This suggests that the C-terminal domain may stabilise the PrD against prion formation by steric means, and implies that the PrD does not induce prion formation by altering the thermodynamic stability of the folded protein. 相似文献
3.
The equilibrium and kinetic folding of hen egg-white lysozyme was studied by means of circular dichroism spectra in the far- and near-ultraviolet (UV) regions at 25 degrees C under the acidic pH conditions. In equilibrium condition at pH 2.2, hen lysozyme shows a single cooperative transition in the GdnCl-induced unfolding experiment. However, in the GdnCl-induced unfolding process at lower pH 0.9, a distinct intermediate state with molten globule characteristics was observed. The time-dependent unfolding and refolding of the protein were induced by concentration jumps of the denaturant and measured by using stopped-flow circular dichroism at pH 2.2. Immediately after the dilution of denaturant, the kinetics of refolding shows evidence of a major unresolved far-UV CD change during the dead time (<10 ms) of the stopped-flow experiment (burst phase). The observed refolding and unfolding curves were both fitted well to a single-exponential function, and the rate constants obtained in the far- and near-UV regions coincided with each other. The dependence on denaturant concentration of amplitudes of burst phase and both rate constants was modeled quantitatively by a sequential three-state mechanism, U<-->I<-->N, in which the burst-phase intermediate (I) in rapid equilibrium with the unfolded state (U) precedes the rate-determining formation of the native state (N). The role of folding intermediate state of hen lysozyme was discussed. 相似文献
4.
To search for folding intermediates, we have examined the folding and unfolding kinetics of wild-type barnase and four representative mutants under a wide range of conditions that span two-state and multi-state kinetics. The choice of mutants and conditions provided in-built controls for artifacts that might distort the interpretation of kinetics, such as the non-linearity of kinetic and equilibrium data with concentration of denaturant. We measured unfolding rate constants over a complete range of denaturant concentration by using by 1H/2H-exchange kinetics under conditions that favour folding, conventional stopped-flow methods at higher denaturant concentrations and continuous flow. Under conditions that favour multi-state kinetics, plots of the rate constants for unfolding against denaturant concentration fitted quantitatively to the equation for three-state kinetics, with a sigmoid component for a change of rate determining step, as did the refolding kinetics. The position of the transition state on the reaction pathway, as measured by solvent exposure (the Tanford beta value) also moved with denaturant concentration, fitting quantitatively to the same equations with a change of rate determining step. The sigmoid behaviour disappeared under conditions that favoured two-state kinetics. Those data combined with direct structural observations and simulation support a minimal reaction pathway for the folding of barnase that involves two detectable folding intermediates. The first intermediate, I(1), is the denatured state under physiological conditions, D(Phys), which has native-like topology, is lower in energy than the random-flight denatured state U and is suggested by molecular dynamics simulation of unfolding to be on-pathway. The second intermediate, I(2), is high energy, and is proven by the change in rate determining step in the unfolding kinetics to be on-pathway. The change in rate determining step in unfolding with structure or environment reflects the change in partitioning of this intermediate to products or starting materials. 相似文献
5.
Proteins constructed from linear arrays of tandem repeats provide a simplified architecture for understanding protein folding. Here, we examine the folding kinetics of the ankyrin repeat domain from the Drosophila Notch receptor, which consists of six folded ankyrin modules and a seventh partly disordered N-terminal ankyrin repeat sequence. Both the refolding and unfolding kinetics are best described as a sum of two exponential phases. The slow, minor refolding phase is limited by prolyl isomerization in the denatured state (D). The minor unfolding phase, which appears as a lag during fluorescence-detected unfolding, is consistent with an on-pathway intermediate (I). This intermediate, although not directly detected during refolding, is shown to be populated by interrupted refolding experiments. When plotted against urea, the rate constants for the major unfolding and refolding phases define a single non-linear v-shaped chevron, as does the minor unfolding phase. These two chevrons, along with unfolding amplitudes, are well-fitted by a sequential three-state model, which yields rate constants for the individual steps in folding and unfolding. Based on these fitted parameters, the D to I step is rate-limiting, and closely matches the major observed refolding phase at low denaturant concentrations. I appears to be midway between N and D in folding free energy and denaturant sensitivity, but has Trp fluorescence properties close to N. Although the Notch ankyrin domain has a simple architecture, folding is slow, with the limiting refolding rate constant as much as seven orders of magnitude smaller than expected from topological predictions. 相似文献
6.
Unfolding and refolding of dimeric creatine kinase equilibrium and kinetic studies. 总被引:2,自引:0,他引:2
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Y. X. Fan J. M. Zhou H. Kihara C. L. Tsou 《Protein science : a publication of the Protein Society》1998,7(12):2631-2641
Equilibrium and kinetic studies of the guanidine hydrochloride induced unfolding-refolding of dimeric cytoplasmic creatine kinase have been monitored by intrinsic fluorescence, far ultraviolet circular dichroism, and 1-anilinonaphthalene-8-sulfonate binding. The GuHCl induced equilibrium-unfolding curve shows two transitions, indicating the presence of at least one stable equilibrium intermediate in GuHCl solutions of moderate concentrations. This intermediate is an inactive monomer with all of the thiol groups exposed. The thermodynamic parameters obtained by analysis using a three-state model indicate that this intermediate is similar in energy to the fully unfolded state. There is a burst phase in the refolding kinetics due to formation of an intermediate within the dead time of mixing (15 ms) in the stopped-flow apparatus. Further refolding to the native state after the burst phase follows biphasic kinetics. The properties of the burst phase and equilibrium intermediates were studied and compared. The results indicate that these intermediates are similar in some respects, but different in others. Both are characterized by pronounced secondary structure, compact globularity, exposed hydrophobic surface area, and the absence of rigid side-chain packing, resembling the "molten globule" state. However, the burst phase intermediate shows more secondary structure, more exposed hydrophobic surface area, and more flexible side-chain packing than the equilibrium intermediate. Following the burst phase, there is a fast phase corresponding to folding of the monomer to a compact conformation. This is followed by rapid assembly to form the dimer. Neither of the equilibrium unfolding transitions are protein concentration dependent. The refolding kinetics are also not concentration dependent. This suggests that association of the subunits is not rate limiting for refolding, and that under equilibrium conditions, dissociation occurs in the region between the two unfolding transitions. Based upon the above results, schemes of unfolding and refolding of creatine kinase are proposed. 相似文献
7.
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
8.
Meng Ge Yong-Jin Mao Xian-Ming Pan 《Extremophiles : life under extreme conditions》2009,13(1):131-137
The α/β-mixed dimeric protein Ssh10b from the hyperthermophile Sulfolobus shibatae is a member of the Sac10b family that is thought to be involved in chromosomal organization or DNA repair/recombination.
The equilibrium unfolding/refolding of Ssh10b induced by denaturants and heat was fully reversible, suggesting that Ssh10b
could serve as a good model for folding/unfolding studies of protein dimers. Here, we investigate the folding/unfolding kinetics
of Ssh10b in detail by stopped-flow circular dichroism (SF-CD) and using GdnHCl as denaturant. In unfolding reactions, the
native Ssh10b turned rapidly into fully unfolded monomers within the stopped-flow dead time with no detectable kinetic intermediate,
agreeing well with the results of equilibrium unfolding experiments. In refolding reactions, two unfolded monomers associate
in the burst phase to form a dimeric intermediate that undergoes a further, slower, first-order folding process to form the
native dimer. Our results demonstrate that the dimerization is essential for maintaining the native tertiary interactions
of the protein Ssh10b. In addition, folding mechanisms of Ssh10b and several other α/β-mixed or pure β-sheet proteins are
compared. 相似文献
9.
The unfolding transition and kinetic refolding of dimeric creatine kinase after urea denaturation were monitored by intrinsic fluorescence and far ultraviolet circular dichroism. An equilibrium intermediate and a kinetic folding intermediate were identified and characterized. The fluorescence intensity of the equilibrium intermediate is close to that of the unfolded state, whereas its ellipticity at 222 nm is about 50% of the native state. The transition curves measured by these two methods are therefore non-coincident. The kinetic folding intermediate, formed during the burst phase of refolding under native-like conditions, possesses 75% of the native secondary structure, but is mostly lacking in native tertiary structure. In moderate concentrations of urea, only the initial, rapid change in fluorescence intensity or negative ellipticity is observed, and the final state values do not reach the equivalent unfolding values. The unfolding and refolding transition curves measured under identical conditions are non-coincident within the transition from intermediate to fully unfolded state. It is observed by SDS-PAGE that disulfide bond-linked dimeric or oligomeric intermediates are formed in moderate urea concentrations, especially in the refolding reaction. These rapidly formed, soluble intermediates represent an off-pathway event that leads to the hysteresis in the refolding transition curves. 相似文献
10.
Multiple phases have been observed during the folding and unfolding of intestinal fatty acid binding protein (WT-IFABP) by stopped-flow fluorescence. Site-directed mutagenesis has been used to examine the role of each of the two tryptophans of this protein in these processes. The unfolding and refolding kinetics of the mutant protein containing only tryptophan 82 (W6Y-IFABP) showed that the tryptophan at this location was critical to the fluorescence signal changes observed throughout the unfolding reaction and early in the refolding reaction. However, the kinetic patterns of the mutant protein containing only tryptophan 6 (W82Y-IFABP) indicated that the tryptophan at this location participated in the fluorescence signal changes observed early in the unfolding reaction and late in the refolding reaction. Together, these data suggest that native-like structure was formed first in the vicinity of tryptophan 82, near the center of the hydrophobic core of this beta-sheet protein, prior to formation of native-like structure in the periphery of the protein. 相似文献
11.
Refolding rate of stability-enhanced cytochrome c is independent of thermodynamic driving force. 总被引:2,自引:2,他引:0
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N52I iso-2 cytochrome c is a variant of yeast iso-2 cytochrome c in which asparagine substitutes for isoleucine 52 in an alpha helical segment composed of residues 49-56. The N52I substitution results in a significant increase in both stability and cooperativity of equilibrium unfolding, and acts as a "global suppressor" of destabilizing mutations. The equilibrium m-value for denaturant-induced unfolding of N52I iso-2 increases by 30%, a surprisingly large amount for a single residue substitution. The folding/unfolding kinetics for N52I iso-2 have been measured by stopped-flow mixing and by manual mixing, and are compared to the kinetics of folding/unfolding of wild-type protein, iso-2 cytochrome c. The results show that the observable folding rate and the guanidine hydrochloride dependence of the folding rate are the same for iso-2 and N52I iso-2, despite the greater thermodynamic stability of N52I iso-2. Thus, there is no linear free-energy relationship between mutation-induced changes in stability and observable refolding rates. However, for N52I iso-2 the unfolding rate is slower and the guanidine hydrochloride dependence of the unfolding rate is smaller than for iso-2. The differences in the denaturant dependence of the unfolding rates suggest that the N52I substitution decreases the change in the solvent accessible hydrophobic surface between the native state and the transition state. Two aspects of the results are inconsistent with a two-state folding/unfolding mechanism and imply the presence of folding intermediates: (1) observable refolding rate constants calculated from the two-state mechanism by combining equilibrium data and unfolding rate measurements deviate from the observed refolding rate constants; (2) kinetically unresolved signal changes ("burst phase") are observed for both N52I iso-2 and iso-2 refolding. The "burst phase" amplitude is larger for N52I iso-2 than for iso-2, suggesting that the intermediates formed during the "burst phase" are stabilized by the N52I substitution. 相似文献
12.
13.
Equilibria and kinetics of folding/unfolding of α-lactalbumin and its two N-terminal variants were studied by circular dichroism spectroscopy. The two variants were wild-type recombinant and Glu1-deletion (E1M) variants expressed in Escherichia coli. The presence of an extra methionine at the N terminus in recombinant α-lactalbumin destabilized the protein by 2 kcal/mol, while the stability was recovered in the E1M variant in which Glu1 was replaced by Met1. Kinetic folding/unfolding reactions of the proteins, induced by stopped-flow concentration jumps of guanidine hydrochloride, indicated the presence of a burst-phase in refolding, and gave chevron plots with significant curvatures in both the folding and unfolding limbs. The folding-limb curvature was interpreted in terms of accumulation of the burst-phase intermediate. However, there was no burst phase observed in the unfolding kinetics to interpret the unfolding-limb curvature. We thus assumed a sequential four-state mechanism, in which the folding from the burst-phase intermediate takes place via two transition states separated by a high-energy intermediate. We estimated changes in the free energies of the burst-phase intermediate and two transition states, caused by the N-terminal variations and also by the presence of stabilizing calcium ions. The Φ values at the N terminus and at the Ca(2+)-binding site thus obtained increased successively during folding, demonstrating the validity of the sequential mechanism. The stability and the folding behavior of the E1M variant were essentially identical to those of the authentic protein, allowing us to use this variant as a pseudo-wild-type α-lactalbumin in future studies. 相似文献
14.
Smajlović A Berbić S Žerovnik E 《Biochemical and biophysical research communications》2011,(2):337-341
The role of the aromatic residue at site 75 to protein stability, the mechanism of folding and the mechanism of amyloid-fibril formation were investigated for the human stefin B variant (bearing Y at site 31) and its point mutation H75W. With an aim to reveal the conformation at the cross-road between folding and aggregation, first, the kinetics of folding and oligomer formation by human stefin B(Y31) variant were studied. It was found to fold in three kinetic phases at pH 4.8 and 10% TFE; the pH and solvent conditions that transform the protein into amyloid fibrils at longer times. The same pH leads to the formation of native-like intermediate (known from previous studies of this variant), meaning that the process of folding and amyloid-fibril formation share the same structural intermediate, which is in this case native-like and dimeric. At pH 5.8 and 7.0 stefin B folded to the native state in four kinetic phases over two intermediates. In distinction, the mutant H75W did not fold to completion, ending in intermediate states at all pH values studied: 4.8, 5.8 and 7.0. At pH 4.8 and 5.8, the mutant folded in one kinetic phase to the intermediate of the “molten globule” type, which leads to the conclusion that its mechanism of folding differs from the one of the parent stefin B at the same pH. At pH 7.0 the mutant H75W folded in three kinetic phases to a native-like intermediate, analogous to folding of stefin B at pH 4.8. 相似文献
15.
Divalent metal cofactor binding in the kinetic folding trajectory of Escherichia coli ribonuclease HI
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Goedken ER Keck JL Berger JM Marqusee S 《Protein science : a publication of the Protein Society》2000,9(10):1914-1921
Proteins often require cofactors to perform their biological functions and must fold in the presence of their cognate ligands. Using circular dichroism spectroscopy. we investigated the effects of divalent metal binding upon the folding pathway of Escherichia coli RNase HI. This enzyme binds divalent metal in its active site, which is proximal to the folding core of RNase HI as defined by hydrogen/deuterium exchange studies. Metal binding increases the apparent stability of native RNase HI chiefly by reducing the unfolding rate. As with the apo-form of the protein, refolding from high denaturant concentrations in the presence of Mg2+ follows three-state kinetics: formation of a rapid burst phase followed by measurable single exponential kinetics. Therefore, the overall folding pathway of RNase HI is minimally perturbed by the presence of metal ions. Our results indicate that the metal cofactor enters the active site pocket only after the enzyme reaches its native fold, and therefore, divalent metal binding stabilizes the protein by decreasing its unfolding rate. Furthermore, the binding of the cofactor is dependent upon a carboxylate critical for activity (Asp10). A mutation in this residue (D10A) alters the folding kinetics in the absence of metal ions such that they are similar to those observed for the unaltered enzyme in the presence of metal. 相似文献
16.
Zhu L Fan YX Perrett S Zhou JM 《Biochemical and biophysical research communications》2001,285(4):857-862
Creatine kinase (CK) is a dimeric enzyme important in ATP regeneration in cells where energy demands are high. The folding of CK under equilibrium and transient conditions has been studied in detail and is found to be complex. At equilibrium in 0.8 M GuHCl, 90% of CK molecules are in the form of a partially structured, monomeric intermediate. We exploit this property to measure kinetics of refolding and unfolding to and from this equilibrium intermediate (EI), using far-UV circular dichroism and intrinsic fluorescence as structural probes. We are thus able to compare the properties of EI and the kinetic intermediate formed during the burst phase in refolding. Native CK and EI unfold with rate constants in seconds and milliseconds, respectively. As is observed for refolding of fully-denatured CK, refolding from EI to the native state shows a burst phase followed by two exponential phases. The burst phase refolding intermediate is inferred to have more structure and greater stability than the equilibrium intermediate. When refolding from the fully-denatured state in 0.8 M GuHCl, the equilibrium intermediate is formed within the dead-time of mixing in the stopped-flow apparatus. The equilibrium intermediate may thus represent a kinetic intermediate formed early during folding. 相似文献
17.
Transmissible spongiform encephalopathies are associated with the conversion of cellular prion protein, PrP(C), into a misfolded oligomeric form, PrP(Sc). Here we have examined the kinetics of folding and unfolding reactions for the recombinant human prion protein C-terminal fragment 90-231 at pH 4.8 and 7.0. The stopped-flow data provide clear evidence for the population of an intermediate on the refolding pathway of the prion protein as indicated by a pronounced curvature in chevron plots and the presence of significant burst phase amplitude in the refolding kinetics. In addition to its role in the normal prion protein folding, this intermediate likely represents a crucial monomeric precursor of the pathogenic PrP(Sc) isoform. 相似文献
18.
Extensive analysis of accurate quench-flow hydrogen exchange results indicates that the burst phase kinetic intermediate in the folding of apomyoglobin (apoMb) from urea is structurally heterogeneous. The structural variability is associated with the partial folding of the E helix during the burst phase (<6.4ms) of the folding process. Analysis of the effects of exchange-out of amide proton labels during the labeling pulse ( approximately pH 10) of the quench-flow process indicates that three of the amide protons in the E helix are in fact largely protected in the burst phase of folding, while the remainder of the E helix has a substantial complement of amide protons that show biphasic kinetics, i.e. are protected partly during the burst phase and partly during the slow phase of folding. The locations of these amide protons can be used to map the sites of structural heterogeneity in the kinetic molten globule. These sites include, besides the E helix, the ends of the A and B helices and part of the C helix. Our results give significant support to the hypothesis that the kinetic molten globule intermediate of apoMb is native-like. 相似文献
19.
FIS, the factor for inversion stimulation, from Escherichia coli and other enteric bacteria, is an interwined alpha-helical homodimer. Size exclusion chromatography and static light scattering measurements demonstrated that FIS is predominately a stable dimer at the concentrations (1-10 microM monomer) and buffer conditions employed in this study. The folding and unfolding of FIS were studied with both equilibrium and kinetic methods by circular dichroism using urea and guanidinium chloride (GdmCl) as the perturbants. The equilibrium folding is reversible and well-described by a two-state folding model, with stabilities at 10 degrees C of 15.2 kcal mol(-1) in urea and 13.5 kcal mol(-1) in GdmCl. The kinetic data are consistent with a two-step folding reaction where the two unfolded monomers associate to a dimeric intermediate within the mixing time for the stopped-flow instrument (<5 ms), and a slower, subsequent folding of the dimeric intermediate to the native dimer. Fits of the burst phase amplitudes as a function of denaturant showed that the free energy for the formation of the dimeric intermediate constitutes the majority of the stability of the folding (9.6 kcal mol(-1) in urea and 10.5 kcal mol(-1) in GdmCl). Folding-to-unfolding double jump kinetic experiments were also performed to monitor the formation of native dimer as a function of folding delay times. The data here demonstrate that the dimeric intermediate is obligatory and on-pathway. The folding mechanism of FIS, when compared to other intertwined, alpha-helical, homodimers, suggests that a transient kinetic dimeric intermediate may be a common feature of the folding of intertwined, segment-swapped, alpha-helical dimers. 相似文献
20.
Baryshnikova EN Melnik BS Finkelstein AV Semisotnov GV Bychkova VE 《Protein science : a publication of the Protein Society》2005,14(10):2658-2667
When considering protein folding with a transient intermediate, a difficulty arises as to determination of the rates of separate transitions. Here we overcome this problem, using the kinetic studies of the unfolding/refolding reactions of the three-state protein apomyoglobin as a model. Amplitudes of the protein refolding kinetic burst phase corresponding to the transition from the unfolded (U) to intermediate (I) state, that occurs prior to the native state (N) formation, allow us to estimate relative populations of the rapidly converting states at various final urea concentrations. On the basis of these proportions, a complicated experimental chevron plot has been deconvolved into the urea-dependent rates of the I<-->N and U<-->N transitions to give the dependence of free energies of the main transition state and of all three (N, I, and U) stable states on urea concentration. 相似文献