Unfolding rates of barstar determined in native and low denaturant conditions indicate the presence of intermediates

The folding and unfolding rates of the small protein, barstar, have been monitored using stopped-flow measurements of intrinsic tryptophan fluorescence at 25 degrees C, pH 8.5, and have been compared over a wide range of urea and guanidine hydrochloride (GdnHCl) concentrations. When the logarithms of the rates of folding from urea and from GdnHCl unfolded forms are extrapolated linearly with denaturant concentration, the same rate is obtained for folding in zero denaturant. Similar linear extrapolations of rates of unfolding in urea and GdnHCl yield, however, different unfolding rates in zero denaturant, indicating that such linear extrapolations are not valid. It has been difficult, for any protein, to determine unfolding rates under nativelike conditions in direct kinetic experiments. Using a novel strategy of coupling the reactivity of a buried cysteine residue with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) to the unfolding reaction of barstar, the global unfolding and refolding rates have now been determined in low denaturant concentrations. The logarithms of unfolding rates obtained at low urea and GdnHCl concentrations show a markedly nonlinear dependence on denaturant concentration and converge to the same unfolding rate in the absence of denaturant. It is shown that the native protein can sample the fully unfolded conformation even in the absence of denaturant. The observed nonlinear dependences of the logarithms of the refolding and unfolding rates observed for both denaturants are shown to be due to the presence of (un)folding intermediates and not due to movements in the position of the transition state with a change in denaturant concentration.     

Thermodynamics of the denaturation of pepsinogen by urea.

The denaturation of swine pepsinogen has been studied as a function of urea concentration, pH, and temperature. The unfolding of the protein by urea has been found to be fully reversible under different conditions of pH, temperature, and denaturant concentration. Kinetic experiments have shown that the transition shows two-state behavior at 25 degrees C in the pH range 6-8 covered in this study. Analysis of the equilibrium data obtained at 25 degrees C according to Tanford (Tanford, C. (1970), Adv. Protein Chem. 24, 1) and Pace (Pace, N.C. (1975), Crit. Rev. Biochem. 3, 1) leads to the conclusion that the free energy of stabilization of native pepsinogen, relative to the denatured state, under physiological conditions, is only 6-12 kcal mol-1. The temperature dependence of the equilibrium constant for the unfolding of pepsinogen by urea in the range 20-50 degrees C at pH 8.0 can be described by assigning the following values of thermodynamic parameters for the denaturation at 25 degrees C: deltaH=31.5 kcal mol-1; deltaS=105 cal deg-1 mol-1; and deltaCp=5215 cal deg-1 mol-1.     

Folding and stability of trp aporepressor from Escherichia coli

Equilibrium and kinetic studies of the urea-induced unfolding of trp aporepressor from Escherichia coli were performed to probe the folding mechanism of this intertwined, dimeric protein. The equilibrium unfolding transitions at pH 7.6 and 25 degrees C monitored by difference absorbance, fluorescence, and circular dichroism spectroscopy are coincident within experimental error. All three transitions are well described by a two-state model involving the native dimer and the unfolded monomer; the free energy of folding in the absence of denaturant and under standard-state conditions is estimated to be 23.3 +/- 0.9 kcal/mol of dimer. The midpoint of the equilibrium unfolding transition increases with increasing protein concentration in the manner expected from the law of mass action for the two-state model. We find no evidence for stable folding intermediates. Kinetic studies reveal that unfolding is governed by a single first-order reaction whose relaxation time decreases exponentially with increasing urea concentration and also decreases with increasing protein concentration in the transition zone. Refolding involves at least three phases that depend on both the protein concentration and the final urea concentration in a complex manner. The relaxation time of the slowest of these refolding phases is identical with that for the single phase in unfolding in the transition zone, consistent with the results expected for a reaction that is kinetically reversible. The two faster refolding phases are presumed to arise from slow isomerization reactions in the unfolded form and reflect parallel folding channels.     

Characterization of the folding and unfolding reactions of single-chain monellin: evidence for multiple intermediates and competing pathways

The mechanisms of folding and unfolding of the small plant protein monellin have been delineated in detail. For this study, a single-chain variant of the natively two-chain monellin, MNEI, was used, in which the C terminus of chain B was connected to the N terminus of chain A by a Gly-Phe linker. Equilibrium guanidine hydrochloride (GdnHCl)-induced unfolding experiments failed to detect any partially folded intermediate that is stable enough to be populated at equilibrium to a significant extent. Kinetic experiments in which the refolding of GdnHCl-unfolded protein was monitored by measurement of the change in the intrinsic tryptophan fluorescence of the protein indicated the accumulation of three transient partially structured folding intermediates. The fluorescence change occurred in three kinetic phases: very fast, fast, and slow. It appears that the fast and slow changes in fluorescence occur on competing folding pathways originating from one unfolded form and that the very fast change in fluorescence occurs on a third parallel pathway originating from a second unfolded form of the protein. Kinetic experiments in which the refolding of alkali-unfolded protein was monitored by the change in the fluorescence of the hydrophobic dye 8-anilino-1-naphthalenesulfonic acid (ANS), consequent to the dye binding to the refolding protein, as well as by the change in intrinsic tryptophan fluorescence, not only confirmed the presence of the three kinetic intermediates but also indicated the accumulation of one or more early intermediates at a few milliseconds of refolding. These experiments also exposed a very slow kinetic phase of refolding, which was silent to any change in the intrinsic tryptophan fluorescence of the protein. Hence, the spectroscopic studies indicated that refolding of single-chain monellin occurs in five distinct kinetic phases. Double-jump, interrupted-folding experiments, in which the accumulation of folding intermediates and native protein during the folding process could be determined quantitatively by an unfolding assay, indicated that the fast phase of fluorescence change corresponds to the accumulation of two intermediates of differing stabilities on competing folding pathways. They also indicated that the very slow kinetic phase of refolding, identified by ANS binding, corresponds to the formation of native protein. Kinetic experiments in which the unfolding of native protein in GdnHCl was monitored by the change in intrinsic tryptophan fluorescence indicated that this change occurs in two kinetic phases. Double-jump, interrupted-unfolding experiments, in which the accumulation of unfolding intermediates and native protein during the unfolding process could be determined quantitatively by a refolding assay, indicated that the fast unfolding phase corresponds to the formation of fully unfolded protein via one unfolding pathway and that the slow unfolding phase corresponds to a separate unfolding pathway populated by partially unfolded intermediates. It is shown that the unfolded form produced by the fast unfolding pathway is the one which gives rise to the very fast folding pathway and that the unfolded form produced by the slower unfolding pathway is the one which gives rise to the slow and fast folding pathways.     

The guanidine like effects of arginine on aminoacylase and salt-induced molten globule state

Aminoacylase is a dimeric enzyme containing one Zn(2+) ion per subunit. The arginine (Arg)-induced unfolding of Holo-aminoacylase and Apo-aminoacylase has been studied by measurement of enzyme activity, fluorescence emission spectra and 1-anilino-8-naphthalenesulfonate (ANS) fluorescence spectra. Besides being the most alkaline amino acid, the arginine molecule contains a positively charged guanidine group, similar to guanidine hydrochloride, and has been used in many refolding systems to suppress protein aggregation. Our results showed that arginine caused the inactivation and unfolding of aminoacylase, with no aggregation during denaturation. A comparison between the unfolding of aminoacylase in aqueous and HCl (pH 7.5) arginine solutions indicated that the guanidine group of arginine had protein-denaturing effects similar to those of guanidine hydrochloride, which might help us understand the mechanism by which arginine suppresses incorrect refolding. The results showed that arginine-denatured aminoacylase could be reactivated and refolded correctly, indicating that arginine is as good a denaturant as the guanidine or urea for study of protein unfolding and refolding. Both the intrinsic fluorescence and the ANS fluorescence spectra showed that the arginine-unfolded aminoacylase formed a molten globule state in the presence of KCl, suggesting that intermediates exist during aminoacylase refolding. The results for the Apo-aminoacylase followed were similar to those for the Holo-enzyme, suggesting that Holo- and Apo-aminoacylase might have a similar unfolding and refolding pathway.     

Folding of dihydrofolate reductase from Escherichia coli

The urea-induced equilibrium unfolding transition of dihydrofolate reductase from Escherichia coli was monitored by UV difference, circular dichroism (CD), and fluorescence spectroscopy. Each of these data sets were well described by a two-state unfolding model involving only native and unfolded forms. The free energy of folding in the absence of urea at pH 7.8, 15 degrees C is 6.13 +/- 0.36 kcal mol-1 by difference UV, 5.32 +/- 0.67 kcal mol-1 by CD, and 5.42 +/- 1.04 kcal mol-1 by fluorescence spectroscopy. The midpoints for the difference UV, CD, and fluorescence transitions are 3.12, 3.08, and 3.18 M urea, respectively. The near-coincidence of the unfolding transitions monitored by these three techniques also supports the assignment of a two-state model for the equilibrium results. Kinetic studies of the unfolding and refolding reactions show that the process is complex and therefore that additional species must be present. Unfolding jumps in the absence of potassium chloride revealed two slow phases which account for all of the amplitude predicted by equilibrium experiments. Unfolding in the presence of 400 mM KCl results in the selective loss of the slower phase, implying that there are two native forms present in equilibrium prior to unfolding. Five reactions were observed in refolding: two slow phases designated tau 1 and tau 2 that correspond to the slow phases in unfolding and three faster reactions designated tau 3, tau 4, and tau 5 that were followed by stopped-flow techniques. The kinetics of the recovery of the native form was monitored by following the binding of methotrexate, a tight-binding inhibitor of dihydrofolate reductase, at 380 nm.(ABSTRACT TRUNCATED AT 250 WORDS)     

Folding-unfolding equilibrium and kinetics of equine beta-lactoglobulin: equivalence between the equilibrium molten globule state and a burst-phase folding intermediate

The denaturant-induced equilibrium unfolding transition of equine beta-lactoglobulin was investigated by ultraviolet absorption, fluorescence, and circular dichroism (CD) spectra. An equilibrium intermediate populates at moderate denaturant concentrations, and its CD spectrum is similar to that of the molten globule state previously observed for this protein at acid pH [Ikeguchi, M., Kato, S., Shimizu, A., and Sugai, S. (1997) Proteins: Struct., Funct., Genet. 27, 567-575]. The unfolding and refolding kinetics were also investigated by the stopped-flow CD and fluorescence. A significant change in the CD intensity was observed within the dead time of measurements (25 ms) when the refolding reaction was initiated by diluting the urea-unfolded protein solution, indicating the transient accumulation of the folding intermediate. The CD spectrum of this burst-phase intermediate agrees well with that of the molten globule state at acid pH. The stability of the burst-phase intermediate was also estimated from the urea-concentration dependence of the burst-phase amplitude, and it shows a fair agreement with that of the equilibrium intermediate. These results indicate that the molten globule state of equine beta-lactoglobulin populates at moderate urea concentration as well as at acid pH and it is equivalent with the kinetic folding intermediate.     

Unassisted refolding of urea-denatured arginine kinase from shrimp Feneropenaeus chinensis: evidence for two equilibrium intermediates in the refolding pathway

The refolding process and the equilibrium intermediates of urea-denatured arginine kinase (AK) were investigated by 1-anilino-8-naphthalenesulfonate (ANS) intrinsic fluorescence, far-UV circular dichroism (CD), size-exclusion chromatography (SEC), and enzymatic activity. In dilute denaturant, two equilibrium refolding intermediates (I and N') were discovered, and a refolding scheme of urea-denatured AK was proposed. During the refolding of urea-denatured AK, the fluorescence intensity increased remarkably, accompanied by a significant blue shift of the emission maximum and a pronounced increase in molar ellipticity of CD at 222 nm. The first folding intermediate (I) was inactive in urea solution ranging between 2.4 and 3.0 M. The second (N') existed between a 0.4- and 0.8-M urea solution, with slightly increased activity. Neither the blue shift emission maximum nor the molar ellipticity of CD at 222 nm showed significant changes in these two regions. The two intermediates were characterized by monitoring the ANS binding ability in various residual urea solutions, and two peaks of the emission intensity were observed in urea solutions of 0.6 and 2.8 M, respectively. The SEC results indicated that a distribution coefficient (K(D)) platform existed in urea solutions ranging between 2.4 and 3.0 M urea, suggesting that there was a similarly apparent protein profile and size in the urea solution region. The refolding kinetics showed that the urea-denatured AK was in two-phase refolding. Proline isomerization occurred in the unfolding process of AK, which blocked the slow phase of refolding. These results suggested that the refolding process of urea-denatured AK contained at the least two equilibrium refolding intermediates.     

Kinetic studies on the unfolding and refolding of pepsinogen in urea. The nature of the rate-limiting step

Kinetic studies on the unfolding of pepsinogen by urea showed that changes in absorbance and potential pepsin activity followed simple first order kinetics. Changes in these variables on refolding were more complex. A large part of the absorbance was recovered within the mixing time of these experiments, whereas the appearance of activity was a slow sigmoidal function of time. The results were interpreted to show that pepsinogen can rapidly regain a globular form, but that its activatable form is produced by a slow conformational change in the folded protein. The enthalpies of activation of this change are similar to those of the cis-trans isomerization of proline residues. If the latter reaction is involved in the folding of pepsinogen, it must occur after extensive folding has already occurred.     

Experimental characterization of the folding kinetics of the notch ankyrin domain

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.     

Equilibrium and kinetic measurements of the conformational transition of thioredoxin in urea

Addition of urea to solutions of Escherichia coli thioredoxin results in a cooperative unfolding of the protein centered at 6.7 M urea at 25 degrees C and 5.1 M urea at 2 degrees C and neutral pH as judged by changes in tryptophan fluorescence emission, far-ultraviolet circular dichroism, and exclusion chromatography. Kinetic profiles of changes in tryptophan fluorescence emission intensity were analyzed following either manual or stopped-flow mixing to initiate unfolding or refolding. Unfolding of the native protein occurs in a single kinetic phase whose time constant is markedly dependent on urea concentration. Refolding of the urea-denatured protein occurs in a multiplicity of kinetic phases whose time constants and fractional amplitudes are also dependent upon urea concentration. Urea gradient gel electrophoretic and exclusion chromatographic measurements suggest the transient accumulation of at least one and likely two compact nativelike intermediate conformations during refolding. Simulations of both electrophoretic and chromatographic results suggest that the intermediate conformations are generated by the concerted action of the middle and fast refolding phases.     

The equilibrium unfolding intermediate observed at pH 4 and its relationship with the kinetic folding intermediates in green fluorescent protein

Folding mechanisms of a variant of green fluorescent protein (F99S/M153T/V163A) were investigated by a wide variety of spectroscopic techniques. Equilibrium measurements on acid-induced denaturation of the protein monitored by chromophore and tryptophan fluorescence and small-angle X-ray scattering revealed that this protein accumulates at least two equilibrium intermediates, a native-like intermediate and an unfolding intermediate, the latter of which exhibits the characteristics of the molten globule state under moderately denaturing conditions at pH 4. To elucidate the role of the equilibrium unfolding intermediate in folding, a series of kinetic refolding experiments with various combinations of initial and final pH values, including pH 7.5 (the native condition), pH 4.0 (the moderately denaturing condition where the unfolding intermediate is accumulated), and pH 2.0 (the acid-denaturing condition) were carried out by monitoring chromophore and tryptophan fluorescence. Kinetic on-pathway intermediates were accumulated during the folding on the refolding reaction from pH 2.0 to 7.5. However, the signal change corresponding to the conversion from the acid-denatured to the kinetic intermediate states was significantly reduced on the refolding reaction from pH 4.0 to pH 7.5, whereas only the signal change corresponding to the above conversion was observed on the refolding reaction from pH 2.0 to pH 4.0. These results indicate that the equilibrium unfolding intermediate is composed of an ensemble of the folding intermediate species accumulated during the folding reaction, and thus support a hierarchical model of protein folding.     

Kinetic studies of the unfolding-refolding of horse muscle phosphoglycerate kinase induced by guanidine hydrochloride

The kinetics of the unfolding and refolding of horse muscle phosphoglycerate kinase were studied with three different signals: fluorescence emission intensity at 336 nm (excitation at 292 nm), ellipticity at 220 nm, and enzyme activity. The results corroborate the conclusion on the existence of intermediates in the folding pathway obtained from equilibrium studies. Kinetic studies showed at least two phases of refolding, as revealed by fluorescence as well as by circular dichroism measurements. During the fast phase, an intermediate was formed with a fluorescence intensity higher than that of the native protein, but devoid of enzyme activity. The fluorescence emission spectrum of this intermediate was determined. Only the slow phase was detected for the unfolding process; it was not attributable to proline isomerization. Several models were assumed, and simulated kinetics derived from these models were compared with the experimental results. A plausible one accounting for most of the data is proposed.     

Mechanism of enhancement of prochymosin renaturation by solubilization of inclusion bodies at alkaline pH

The renaturation efficiency of recombinant prochymosin depends on not only the renaturation condi-tions but also the solubilization (denaturation) conditions. Compared with pH 8, solubilization of prochymosin-contain-ing inclusion bodies at pH 11 (8 mol/L urea) results in onefold increase of renaturation efficiency ( ~ 40% vs. ~ 20 % ). Alkaline pH facilitates the solubilization of inclusion bodies via the breakage of intermolecular disulfide bonds. Moreover, alkaline pH renders prochymosin molecules to be in a more reduced and more unfolded state which undergoes refolding readily.     

绿脓杆菌apoazurin在溶液中存在两种天然构象

如何解释绿脓杆菌apoazurin变性过程的复杂机制是一个有争议的问题.最近的研究表明apoazurin的复杂变性机制可以归结为其天然态存在着至少两种构象.利用内源荧光发射谱和圆二色谱进一步研究了apoazurin的脲变性机制,发现其稳态脲变性符合表观的二态过程,但其动力学为双相过程.在高浓度脲中快反应在几秒钟内完成,而慢反应要经过几个小时.快反应和慢反应的mu值分别为2.24和2.45kJ·mol-1·M-1,去折叠活化能的差值为22kJ·mol-1.时间分辨的荧光发射谱和圆二色谱可以用天然态和完全变性态的谱图通过一个固定的比例参数进行重建.结果表明,过去被广泛接受的存在着变性中间体的机制是不正确的,而apoazurin在天然态存在至少两种构象的假设是合理的.     

Observation of multistate kinetics during the slow folding and unfolding of barstar.

The kinetics of the slow folding and unfolding reactions of barstar, a bacterial ribonuclease inhibitor protein, have been studied at 23(+/-1) degrees C, pH 8, by the use of tryptophan fluorescence, far-UV circular dichroism (CD), near-UV CD, and transient mixing (1)H nuclear magnetic resonance (NMR) spectroscopic measurements in the 0-4 M range of guanidine hydrochloride (GdnHCl) concentration. The denaturant dependences of the rates of folding and unfolding processes, and of the initial and final values of optical signals associated with these kinetic processes, have been determined for each of the four probes of measurement. Values determined for rates as well as amplitudes are shown to be very much probe dependent. Significant differences in the intensities and rates of appearance and disappearance of several resolved resonances in the real-time one-dimensional NMR spectra have been noted. The NMR spectra also show increasing dispersion of chemical shifts during the slow phase of refolding. The denaturant dependences of rates display characteristic folding chevrons with distinct rollovers under strongly native as well as strongly unfolding conditions. Analyses of the data and comparison of the results obtained with different probes of measurement appear to indicate the accumulation of a myriad of intermediates on parallel folding and unfolding pathways, and suggest the existence of an ensemble of transition states. The energetic stabilities of the intermediates estimated from kinetic data suggest that they are approximately half as stable as the fully folded protein. The slowness of the folding and unfolding processes (tau = 10-333 s) and values of 20.5 (+/-1.4) and 18 (+/-0.5) kcal mol(-)(1) for the activation energies of the slow refolding and unfolding reactions suggest that proline isomerization is involved in these reactions, and that the intermediates accumulate and are therefore detectable because the slow proline isomerization reaction serves as a kinetic trap during folding.     

Analysis of folding and unfolding reactions of cytochrome b5

The guanidine hydrochloride- (GuHCl-) induced unfolding and refolding of a recombinant domain of bovine microsomal cytochrome b(5) containing the first 104 amino acid residues has been characterized by both transient and equilibrium spectrophotometric methods. The soluble domain is reversibly unfolded and the equilibrium reaction may be monitored by changes in absorbance and fluorescence that accompany denaturation of the native protein. Both probes reveal a single cooperative transition with a midpoint at 3 M GuHCl and lead to a value for the protein stability (DeltaG(uw)) of 26.5 kJ mol(-1). This stability is much higher than that reported for the corresponding form of the apoprotein (approximately 7 kJ mol(-1)). Transient changes in fluorescence and absorbance during protein unfolding exhibit biphasic profiles. A fast phase occupying approximately 30% of the total amplitude is observed at high denaturant concentrations and becomes the dominant process within the transition region. The rates associated with each process show a linear dependency on GuHCl concentration, and at zero denaturant concentration the unfolding rates (k(uw)) are 4.5 x 10(-5) s(-1) and 5.2 x 10(-6) s(-1) at 25 degrees C. The pattern of unfolding is not correlated with covalent heterogeneity, since a wide range of variants and site-directed mutants exhibit identical profiles, nor is the unfolding correlated with cis-trans Pro isomerization in the native state. In comparison with the apo form of cytochrome b(5), the kinetics of refolding and unfolding are more complex and exhibit very different transition states. The data support a model for unfolding in which heme-protein interactions give rise to two discernible rates of unfolding. From an analysis of the activation parameters associated with each process it is established that two structurally similar transition states differing by less than 5 kJ mol(-1) exist in the unfolding reaction. Protein refolding exhibits monophasic kinetics but with distinct curvature apparent in plots of ln k(obs) versus denaturant concentration. The data are interpreted in terms of alternative routes for protein folding in which a "fast track" leads to the rapid ordering of structure around Trp26 for refolding while a slower route requires additional reorganization around the hydrophobic core.     

Equilibrium denaturation studies of mouse beta-nerve growth factor.

Equilibrium denaturation of dimeric mouse beta-nerve growth factor (beta-NGF) has been studied by monitoring changes in the protein's spectroscopic characteristics. Denaturation of beta-NGF in guanidine hydrochloride and urea resulted in an altered intrinsic fluorescence emission spectrum, fluorescence depolarization, and diminished negative circular dichroism. Native-like spectroscopic properties and specific biological activity are restored when denaturant is diluted from unfolded samples, demonstrating that this process is fully reversible. However, refolding of denatured beta-NGF is dependent on the three disulfide bonds present in the native protein and does not readily occur when the disulfide bonds are reduced. Graphical analysis and nonlinear least-squares fitting of beta-NGF denaturation data demonstrate that denaturation is dependent on the concentration of beta-NGF and is consistent with a two-state model involving native dimer and denatured monomer (N2 = 2D). The conformational stability of mouse beta-NGF calculated according to this model is 19.3 +/- 1.1 kcal/mol in 100 mM sodium phosphate at pH 7. Increasing the hydrogen ion concentration resulted in a 25% decrease in beta-NGF stability at pH 4 relative to pH 7.     

Exploring Early Stages of the Chemical Unfolding of Proteins at the Proteome Scale

After decades of using urea as denaturant, the kinetic role of this molecule in the unfolding process is still undefined: does urea actively induce protein unfolding or passively stabilize the unfolded state? By analyzing a set of 30 proteins (representative of all native folds) through extensive molecular dynamics simulations in denaturant (using a range of force-fields), we derived robust rules for urea unfolding that are valid at the proteome level. Irrespective of the protein fold, presence or absence of disulphide bridges, and secondary structure composition, urea concentrates in the first solvation shell of quasi-native proteins, but with a density lower than that of the fully unfolded state. The presence of urea does not alter the spontaneous vibration pattern of proteins. In fact, it reduces the magnitude of such vibrations, leading to a counterintuitive slow down of the atomic-motions that opposes unfolding. Urea stickiness and slow diffusion is, however, crucial for unfolding. Long residence urea molecules placed around the hydrophobic core are crucial to stabilize partially open structures generated by thermal fluctuations. Our simulations indicate that although urea does not favor the formation of partially open microstates, it is not a mere spectator of unfolding that simply displaces to the right of the folded←→unfolded equilibrium. On the contrary, urea actively favors unfolding: it selects and stabilizes partially unfolded microstates, slowly driving the protein conformational ensemble far from the native one and also from the conformations sampled during thermal unfolding.     

Unassisted refolding of urea unfolded rhodanese

In vitro refolding after urea unfolding of the enzyme rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) normally requires the assistance of detergents or chaperonin proteins. No efficient, unassisted, reversible unfolding/folding transition has been demonstrated to date. The detergents or the chaperonin proteins have been proposed to stabilize folding intermediates that kinetically limit folding by aggregating. Based on this hypothesis, we have investigated a number of experimental conditions and have developed a protocol for refolding, without assistants, that gives evidence of a reversible unfolding transition and leads to greater than 80% recovery of native enzyme. In addition to low protein concentration (10 micrograms/ml), low temperatures are required to maximize refolding. Otherwise optimal conditions give less than 10% refolding at 37 degrees C, whereas at 10 degrees C the recovery approaches 80%. The unfolding/refolding phases of the transition curves are most similar in the region of the transition, and refolding yields are significantly reduced when unfolded rhodanese is diluted to low urea concentrations, rather than to concentrations near the transition region. This is consistent with the formation of "sticky" intermediates that can remain soluble close to the transition region. Apparently, nonnative structures, e.g. aggregates, can form rapidly at low denaturant concentrations, and their subsequent conversion to the native structure is slow.