Early events during folding of wild-type staphylococcal nuclease and a single-tryptophan variant studied by ultrarapid mixing

A continuous-flow mixing device with a dead time of 100 micros coupled with intrinsic tryptophan and 1-anilinonaphthalene-8-sulfonate (ANS) fluorescence was used to monitor structure formation during early stages of the folding of staphylococcal nuclease (SNase). A variant with a unique tryptophan fluorophore in the N-terminal beta-barrel domain (Trp76 SNase) was obtained by replacing the single Trp140 in wild-type SNase with His in combination with Trp substitution of Phe76. A common background of P47G, P117G and H124L mutations was chosen in order to stabilize the protein and prevent accumulation of cis proline isomers under native conditions. In contrast to WT(*) SNase, which shows no changes in tryptophan fluorescence prior to the rate-limiting folding step ( approximately 100 ms), the F76W/W140H variant shows additional changes (enhancement) during an early folding phase with a time constant of 75 micros. Both proteins exhibit a major increase in ANS fluorescence and identical rates for this early folding event. These findings are consistent with the rapid accumulation of an ensemble of states containing a loosely packed hydrophobic core involving primarily the beta-barrel domain while the specific interactions in the alpha-helical domain involving Trp140 are formed only during the final stages of folding. The fact that both variants exhibit the same number of kinetic phases with very similar rates confirms that the folding mechanism is not perturbed by the F76W/W140H mutations. However, the Trp at position 76 reports on the rapid formation of a hydrophobic cluster in the N-terminal beta-sheet region while the wild-type Trp140 is silent during this early stage of folding. Quantitative modeling of the (un)folding kinetics and thermodynamics of these two proteins versus urea concentration revealed that the F76W/W140H mutation selectively destabilizes the native state relative to WT(*) SNase while the stability of transient intermediates remains unchanged, leading to accumulation of intermediates under equilibrium conditions at moderate denaturant concentrations.     

The role of Trp-82 in the folding of intestinal fatty acid binding protein

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.     

Compact dimension of denatured states of staphylococcal nuclease

Fluorescence and circular dichroism stopped-flow have been widely used to determine the kinetics of protein folding including folding rates and possible folding pathways. Yet, these measurements are not able to provide spatial information of protein folding/unfolding. Especially, conformations of denatured states cannot be elaborated in detail. In this study, we apply the method of fluorescence energy transfer with a stopped-flow technique to study global structural changes of the staphylococcal nuclease (SNase) mutant K45C, where lysine 45 is replaced by cysteine, during folding and unfolding. By labeling the thiol group of cysteine with TNB (5,5'-dithiobis-2-nitrobenzoic acid) as an energy acceptor and the tryptophan at position 140 as a donor, distance changes between the acceptor and the donor during folding and unfolding are measured from the efficiency of energy transfer. Results indicate that the denatured states of SNase are highly compact regardless of how the denatured states (pH-induced or GdmCl-induced) are induced. The range of distance changes between two probes is between 25.6 and 25.4 A while it is 20.4 A for the native state. Furthermore, the folding process consists of three kinetic phases while the unfolding process is a single phase. These observations agree with our previous sequential model: N(0) left arrow over right arrow D(1) left arrow over right arrow D(2) left arrow over right arrow D(3) (Chen et al., J Mol Biol 1991;220:771-778). The efficiency of protein folding may be attributed to initiating the folding process from these compact denatured structures.     

pH-induced folding/unfolding of staphylococcal nuclease: determination of kinetic parameters by the sequential-jump method.

On the basis of previous stopped-flow pH-jump experiments, we have proposed that the acid- and alkaline-induced folding/unfolding transition of staphylococcal nuclease, in the time range 2 ms to 300 s, follows the pathway N0 in equilibrium with D1 in equilibrium with D2 in equilibrium with D3, in which D1, D2, and D3 are three substates of the unfolded state and N0 is the native state. The stopped-flow "double-jump" technique has been employed to test this mechanism and to determine the rate constants which would not be accessible by the direct pH jump because of the lack of fluorescence signal, i.e., the rates for the conversion of D1 to D2 and of D2 to D3. In the forward jump, a protein solution kept at pH 7.0 was mixed with an acidic or alkaline solution to the final pH of 3.0 or 12.2, respectively. The mixed solution was kept for varying periods of time, called the delay time, tD. A second mixing (the back jump) was launched to bring the protein solution back to pH 7.0. The time course of the Trp-140 fluorescence signals recovered in the back jump was analyzed as a function of tD. Kinetics of the unfolding were found to be triphasic by the double-jump method, contrary to the monophasic kinetics observed by the direct pH jump. Complex kinetics of unfolding are expected with the proposed kinetic scheme.(ABSTRACT TRUNCATED AT 250 WORDS)     

pH-jump-induced folding and unfolding studies of barstar: evidence for multiple folding and unfolding pathways.

Equilibrium and kinetic characterization of the high pH-induced unfolding transition of the small protein barstar have been carried out in the pH range 7-12. A mutant form of barstar, containing a single tryptophan, Trp 53, completely buried in the core of the native protein, has been used. It is shown that the protein undergoes reversible unfolding above pH 10. The pH 12 form (the D form) appears to be as unfolded as the form unfolded by 6 M guanidine hydrochloride (GdnHCl) at pH 7 (the U form): both forms have similar fluorescence and far-UV circular dichroism (CD) signals and have similar sizes, as determined by dynamic light scattering and size-exclusion chromatography. No residual structure is detected in the D form: addition of GdnHCl does not alter its fluorescence and far-UV CD properties. The fluorescence signal of Trp 53 has been used to monitor folding and unfolding kinetics. The kinetics of folding of the D form in the pH range 7-11 are complex and are described by four exponential processes, as are the kinetics of unfolding of the native state (N state) in the pH range 10.5-12. Each kinetic phase of folding decreases in rate with increase in pH from 7 to 10.85, and each kinetic phase of unfolding decreases in rate with decrease in pH from 12 to 10.85. At pH 10.85, the folding and unfolding rates for any particular kinetic phase are identical and minimal. The two slowest phases of folding and unfolding have identical kinetics whether measured by Trp 53 fluorescence or by mean residue ellipticity at 222 nm. Direct determination of the increase in the N state with time of folding at pH 7 and of the D form with time of unfolding at pH 12, by means of double-jump assays, show that between 85 and 95% of protein molecules fold or unfold via fast pathways between the two forms. The remaining 5-15% of protein molecules appear to fold or unfold via slower pathways, on which at least two intermediates accumulate. The mechanism of folding from the high pH-denatured D form is remarkably similar to the mechanism of folding from the urea or GdnHCl-denatured U form.     

Nonuniform chain collapse during early stages of staphylococcal nuclease folding detected by fluorescence resonance energy transfer and ultrarapid mixing methods

The development of tertiary structure during folding of staphylococcal nuclease (SNase) was studied by time‐resolved fluorescence resonance energy transfer measured using continuous‐ and stopped‐flow techniques. Variants of this two‐domain protein containing intradomain and interdomain fluorescence donor/acceptor pairs (Trp and Cys‐linked fluorophore or quencher) were prepared to probe the intradomain and interdomain structural evolution accompanying SNase folding. The intra‐domain donor/acceptor pairs are within the β‐barrel domain (Trp27/Cys64 and Trp27/Cys97) and the interdomain pair is between the α‐helical domain and the β‐barrel domain (Trp140/Cys64). Time‐resolved energy transfer efficiency accompanying folding and unfolding at different urea concentrations was measured over a time range from 30 μs to ～10 s. Information on average donor/acceptor distances at different stages of the folding process was obtained by using a quantitative kinetic modeling approach. The average distance for the donor/acceptor pairs in the β‐barrel domain decreases to nearly native values whereas that of the interdomain donor/acceptor pairs remains unchanged in the earliest intermediate (<500 μs of refolding). This indicates a rapid nonuniform collapse resulting in an ensemble of heterogeneous conformations in which the central region of the β‐barrel domain is well developed while the C‐terminal α‐helical domain remains disordered. The distance between Trp140 and Cys64 decreases to native values on the 100‐ms time scale, indicating that the α‐helical domain docks onto the preformed β‐barrel at a late stage of the folding. In addition, the unfolded state is found to be more compact under native conditions, suggesting that changes in solvent conditions may induce a nonspecific hydrophobic collapse.     

Two peptide fragments G55-I72 and K97-A109 from staphylococcal nuclease exhibit different behaviors in conformational preferences for helix formation

Two synthetic peptides, SNasealpha1 and SNasealpha2, corresponding to residues G55-I72 and K97-A109, respectively, of staphylococcal nuclease (SNase), are adopted for detecting the role of helix alpha1 (E57-A69) and helix alpha2 (M98-Q106) in the initiation of folding of SNase. The helix-forming tendencies of the two SNase peptide fragments are investigated using circular dichroism (CD) and two-dimensional (2D) nuclear magnetic resonance (NMR) methods in water and 40% trifluoroethanol (TFE) solutions. The coil-helix conformational transitions of the two peptides in the TFE-H2O mixture are different from each other. SNasealpha1 adopts a low population of localized helical conformation in water, and shows a gradual transition to helical conformation with increasing concentrations of TFE. SNasealpha2 is essentially unstructured in water, but undergoes a cooperative transition to a predominantly helical conformation at high TFE concentrations. Using the NMR data obtained in the presence of 40% TFE, an ensemble of alpha-helical structures has been calculated for both peptides in the absence of tertiary interactions. Analysis of all the experimental data available indicates that formation of ordered alpha-helical structures in the segments E57-A69 and M98-Q106 of SNase may require nonlocal interactions through transient contact with hydrophobic residues in other parts of the protein to stabilize the helical conformations in the folding. The folding of helix alpha1 is supposed to be effective in initiating protein folding. The formation of helix alpha2 depends strongly on the hydrophobic environment created in the protein folding, and is more important in the stabilization of the tertiary conformation of SNase.     

Protein Folding Mechanism of the Dimeric AmphiphysinII/Bin1 N-BAR Domain

The human AmphyphisinII/Bin1 N-BAR domain belongs to the BAR domain superfamily, whose members sense and generate membrane curvatures. The N-BAR domain is a 57 kDa homodimeric protein comprising a six helix bundle. Here we report the protein folding mechanism of this protein as a representative of this protein superfamily. The concentration dependent thermodynamic stability was studied by urea equilibrium transition curves followed by fluorescence and far-UV CD spectroscopy. Kinetic unfolding and refolding experiments, including rapid double and triple mixing techniques, allowed to unravel the complex folding behavior of N-BAR. The equilibrium unfolding transition curve can be described by a two-state process, while the folding kinetics show four refolding phases, an additional burst reaction and two unfolding phases. All fast refolding phases show a rollover in the chevron plot but only one of these phases depends on the protein concentration reporting the dimerization step. Secondary structure formation occurs during the three fast refolding phases. The slowest phase can be assigned to a proline isomerization. All kinetic experiments were also followed by fluorescence anisotropy detection to verify the assignment of the dimerization step to the respective folding phase. Based on these experiments we propose for N-BAR two parallel folding pathways towards the homodimeric native state depending on the proline conformation in the unfolded state.     

Folding kinetics and structure of OEP16

The chloroplast outer membrane contains different, specialized pores that are involved in highly specific traffic processes from the cytosol into the chloroplast and vice versa. One representative member of these channels is the outer envelope protein 16 (OEP16), a cation-selective high conductance channel with high selectivity for amino acids. Here we study the mechanism and kinetics of the folding of this membrane protein by fluorescence and circular dichroism spectroscopy, using deletion mutants of the two single tryptophanes Trp-77-->Phe-77 and Trp-100-->Phe-100. In addition, the wild-type spectra were deconvoluted, depicting the individual contributions from each of the two tryptophan residues. The results show that both tryptophan residues are located in a completely different environment. The Trp-77 is deeply buried in the hydrophobic part of the protein, whereas the Trp-100 is partially solvent exposed. These results were further confirmed by studies of fluorescence quenching with I(-). The kinetics of the protein folding are studied by stopped flow fluorescence and circular dichroism measurements. The folding process depends highly on the detergent concentration and can be divided into an ultrafast phase (k > 1000 s(-1)), a fast phase (200-800 s(-1)), and a slow phase (25-70 s(-1)). The slow phase is absent in the W100F mutant. Secondary structure analysis and comparision with closely related proteins led to a new model of the structure of OEP16, suggesting that the protein is, in contrast to most other outer membrane proteins studied so far, purely alpha-helical, consisting of four transmembrane helices. Trp-77 is located in helix II, whereas the Trp-100 is located in the loop between helices II and III, close to the interface to helix III. We suggest that the first, very fast process corresponds to the formation of the helices, whereas the insertion of the helices into the detergent micelle and the correct folding of the II-III loop may be the later, rate-limiting steps of the folding process.     

The role of tryptophan in staphylococcal nuclease stability

Staphylococcal nuclease (SNase) has a single Trp residue at position 140. Circular dichroism, intrinsic and ANS-binding fluorescence, chemical titrations and enzymatic assays were used to measure the changes of its structure, stability and activities as the Trp was mutated or replaced to other positions. The results show that W140 is critical to SNase structure, stability, and function. Mutants such as W140A, F61W/W140A, and Y93W/W140A have unfolding, corrupted secondary and tertiary structures, diminished structural stability and attenuated catalytic activity as compared to the wild type. The deleterious effects of W140 substitution cannot be compensated by concurrent changes at topographical locations of position 61 or 93. Local hydrophobicity defined as a sum of hydrophobicity around a given residue within a distance is found to be a relevant property to SNase folding and stability.     

Domain-domain interface packing at conserved Trp-20 in class alpha glutathione transferase impacts on protein stability

The folding and assembly of the dimeric glutathione transferases (GST) involves the association of two structurally distinct domains per subunit. A prominent and conserved domain-domain interaction in class alpha GSTs is formed by the packing of the indole side chain of Trp-20 from domain I into a hydrophobic pocket in domain II. Stability studies have shown that partial dissociation of the domains near Trp-20 occurs as an initial fast event during the unfolding kinetics of human GSTA1-1 (Wallace et al., Biochemistry 37 (1998) 5320-5328; Wallace et al., Biochem. J. 336 (1998) 413-418). The contribution of Trp-20 toward stabilising the domain-domain interface was investigated by mutating it to either a phenylalanine (W20F) or alanine (W20A) and determining the functionality (catalysis and non-substrate ligand binding) and stability (thermal- and urea-induced denaturation) of the mutant proteins. The replacement of Trp-20 did not impact on the protein's gross structural properties. Functionally, the W20F was non-disruptive, whereas the cavity-creating W20A mutation was. Both mutants destabilised the native state with W20A exerting the greatest effect. Reduced m-values as well as the protein concentration dependence of the urea unfolding transitions for W20F GSTA1-1 suggest the presence of a dimeric intermediate at equilibrium that is not observed with wild-type protein. Unfolding kinetics monitored by stopped-flow tyrosine fluorescence was mono-exponential and corresponded to the global unfolding of the protein during which the dimeric intermediate unfolds to two unfolded monomers. The similar unfolding kinetics data for wild-type and W20F A1-1 indicates that the global unfolding event was not affected by amino acid replacement. We propose that the packing interactions at the conserved Trp-20 plays an important role in stabilising the intrasubunit domain I-domain II interface of class alpha GSTs.     

Core formation in apomyoglobin: probing the upper reaches of the folding energy landscape

An acid-destabilized form of apomyoglobin, the so-called E state, consists of a set of heterogeneous structures that are all characterized by a stable hydrophobic core composed of 30-40 residues at the intersection of the A, G, and H helices of the protein, with little other secondary structure and no other tertiary structure. Relaxation kinetics studies were carried out to characterize the dynamics of core melting and formation in this protein. The unfolding and/or refolding response is induced by a laser-induced temperature jump between the folded and unfolded forms of E, and structural changes are monitored using the infrared amide I' absorbance at 1648-1651 cm(-1) that reports on the formation of solvent-protected, native-like helix in the core and by fluorescence emission changes from apomyoglobin's Trp14, a measure of burial of the indole group of this residue. The fluorescence kinetics data are monoexponential with a relaxation time of 14 micros. However, infrared kinetics data are best fit to a biexponential function with relaxation times of 14 and 59 micros. These relaxation times are very fast, close to the limits placed on folding reactions by diffusion. The 14 micros relaxation time is weakly temperature dependent and thus represents a pathway that is energetically downhill. The appearance of this relaxation time in both the fluorescence and infrared measurements indicates that this folding event proceeds by a concomitant formation of compact secondary and tertiary structures. The 59 micros relaxation time is much more strongly temperature dependent and has no fluorescence counterpart, indicating an activated process with a large energy barrier wherein nonspecific hydrophobic interactions between helix A and the G and H helices cause some helix burial but Trp14 remains solvent exposed. These results are best fit by a multiple-pathway kinetic model when U collapses to form the various folded core structures of E. Thus, the results suggest very robust dynamics for core formation involving multiple folding pathways and provide significant insight into the primary processes of protein folding.     

Transient intermediates in the folding of dihydrofolate reductase as detected by far-ultraviolet circular dichroism spectroscopy

The kinetics of the reversible folding and unfolding of Escherichia coli dihydrofolate reductase have been studied by stopped-flow circular dichroism in the peptide region at pH 7.8 and 15 degrees C. The reactions were induced by concentration jumps of a denaturant, urea. The method can detect various intermediates transiently populated in the reactions although the equilibrium unfolding of the protein is apparently approximated by a two-state reaction. The results can be summarized as follows. (1) From transient circular dichroism spectra measured as soon as the refolding is started, a substantial amount of secondary structure is formed in the burst phase, i.e., within the dead time of stopped-flow mixing (18 ms). (2) The kinetics from this burst-phase intermediate to the native state are multiphasic, consisting of five phases designated as tau 1, tau 2, tau 3, tau 4, and tau 5 in increasing order of the reaction rate. Measurements of the kinetics at various wavelengths have provided kinetic difference circular dichroism spectra for the individual phases. (3) The tau 5 phase shows a kinetic difference spectrum consistent with an exciton contribution of two aromatic residues in the peptide CD region. The absence of the tau 5 phase in a mutant protein, in which Trp 74 is replaced by leucine, suggests that Trp 74 is involved in the exciton pair and that the tau 5 phase reflects the formation of a hydrophobic cluster around Trp 74. From the similarity of the kinetic difference spectrum to the difference between the native spectra of the mutant and wild-type proteins, it appears that Trp 47 is the partner in the exciton pair and that the structure formed in the tau 5 phase persists during the later stages of folding. (4) The later stages of folding show kinetic difference spectra that can be interpreted by rearrangement of secondary structure, particularly the central beta sheet of the protein. The pairwise similarities in the spectrum between the tau 3 and tau 4 phases, and between the tau 1 and tau 2 phases, also suggest the presence of two parallel folding channels for refolding. (5) The unfolding kinetics show three to four phases and are interpreted in terms of the presence of multiple native species. The total ellipticity change in kinetic unfolding reaction, however, agrees with the ellipticity difference between the native and unfolding states, indicating the absence of the burst phase in unfolding.(ABSTRACT TRUNCATED AT 400 WORDS)     

Phage P22 tailspike protein: removal of head-binding domain unmasks effects of folding mutations on native-state thermal stability.

A shortened, recombinant protein comprising residues 109-666 of the tailspike endorhamnosidase of Salmonella phage P22 was purified from Escherichia coli and crystallized. Like the full-length tailspike, the protein lacking the amino-terminal head-binding domain is an SDS-resistant, thermostable trimer. Its fluorescence and circular dichroism spectra indicate native structure. Oligosaccharide binding and endoglycosidase activities of both proteins are identical. A number of tailspike folding mutants have been obtained previously in a genetic approach to protein folding. Two temperature-sensitive-folding (tsf) mutations and the four known global second-site suppressor (su) mutations were introduced into the shortened protein and found to reduce or increase folding yields at high temperature. The mutational effects on folding yields and subunit folding kinetics parallel those observed with the full-length protein. They mirror the in vivo phenotypes and are consistent with the substitutions altering the stability of thermolabile folding intermediates. Because full-length and shortened tailspikes aggregate upon thermal denaturation, and their denaturant-induced unfolding displays hysteresis, kinetics of thermal unfolding were measured to assess the stability of the native proteins. Unfolding of the shortened wild-type protein in the presence of 2% SDS at 71 degrees C occurs at a rate of 9.2 x 10(-4) s(-1). It reflects the second kinetic phase of unfolding of the full-length protein. All six mutations were found to affect the thermal stability of the native protein. Both tsf mutations accelerate thermal unfolding about 10-fold. Two of the su mutations retard thermal unfolding up to 5-fold, while the remaining two mutations accelerate unfolding up to 5-fold. The mutational effects can be rationalized on the background of the recently determined crystal structure of the protein.     

Folding mechanism of the (H3-H4)2 histone tetramer of the core nucleosome

To further understand oligomeric protein assembly, the folding and unfolding kinetics of the H3-H4 histone tetramer have been examined. The tetramer is the central protein component of the core nucleosome, which is the basic unit of DNA compaction into chromatin in the eukaryotic nucleus. This report provides the first kinetic folding studies of a protein containing the histone fold dimerization motif, a motif observed in several protein-DNA complexes. Previous equilibrium unfolding studies have demonstrated that, under physiological conditions, there is a dynamic equilibrium between the H3-H4 dimer and tetramer species. This equilibrium is shifted predominantly toward the tetramer in the presence of the organic osmolyte trimethylamine-N-oxide (TMAO). Stopped-flow methods, monitoring intrinsic tyrosine fluorescence and far-UV circular dichroism, have been used to measure folding and unfolding kinetics as a function of guanidinium hydrochloride (GdnHCl) and monomer concentrations, in 0 and 1 M TMAO. The assignment of the kinetic phases was aided by the study of an obligate H3-H4 dimer, using the H3 mutant, C110E, which destabilizes the H3-H3' hydrophobic four-helix bundle tetramer interface. The proposed kinetic folding mechanism of the H3-H4 system is a sequential process. Unfolded H3 and H4 monomers associate in a burst phase reaction to form a dimeric intermediate that undergoes a further, first-order folding process to form the native dimer in the rate-limiting step of the folding pathway. H3-H4 dimers then rapidly associate with a rate constant of > or =10(7) M(-1)sec(-1) to establish a dynamic equilibrium between the fully assembled tetramer and folded H3-H4 dimers.     

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 analysis of the acid and the alkaline unfolded states of staphylococcal nuclease

Thermodynamic analysis by differential scanning calorimetry shows that the folding/unfolding transition of staphylococcal nuclease is consistent with the two-state process. Stopped-flow kinetic measurements, monitoring the Trp140 fluorescence and covering five decades in time (2 ms to 300 s), indicate that the unfolding from pH 7.0 to 3.1 is monophasic (time constant 1.15 s) and from pH 7.0 to 12.2 is biphasic (time constants: one less than 2 ms and the other 0.6 s). However, the folding, either from pH 3.1 to 7.0 or from pH 12.2 to 7.0, is triphasic (time constants 150 ms, 850 ms and 30 s from acid, 90 ms, 565 ms and 33 s from alkaline). A simple sequential model, which agrees with the above observations for acidic folding/unfolding is, D3 in equilibrium D2 in equilibrium D1 in equilibrium N. The three Ds denote three sub-states of the unfolded state and N denotes the native state. These sub-states of D have similar enthalpy and tryptophan fluorescence, and their equilibrium cannot be shifted by temperature changes. However, they are kinetically distinctive. Data do not favor alternative mechanisms assuming parallel transitions of the three Ds to N, or complexity of the N state, or parallel transitions of sub-states of N1, N2 and N3 to D. Other more complex, branched or cyclic, kinetics are not considered because of the lack of evidence, pH dependence of the unfolding kinetics suggests that the unfolding is triggered by protonation of 0.8(+/- 0.3) ionizable groups, with a pKa of 3.9 or by deprotonation of 1.6(+/- 0.4) ionizable groups with pKa values near 10.5. Circular dichroisms indicate that these three D states retain nonrandom chain conformation. Possible role of these "chain conformation" in the protein folding is discussed.     

Pressure-induced unfolding/refolding of ribonuclease A: static and kinetic Fourier transform infrared spectroscopy study

In this paper, we illustrate the use of high-pressure Fourier transform infrared (FT-IR) spectroscopy to study the reversible presssure-induced unfolding and refolding of ribonuclease A (RNase A) and compare it with the results obtained for the temperature-induced transition. FT-IR spectroscopy monitors changes in the secondary structural properties (amide I' band) or tertiary contacts (tyrosine band) of the protein upon pressurization or depressurization. Analysis of the amide I' spectral components reveals that the pressure-induced denaturation process sets in at 5. 5 kbar at 20 degrees C and pH 2.5. It is accompanied by an increase in disordered structures while the content of beta-sheets and alpha-helices drastically decreases. The denatured state above 7 kbar retains nonetheless some degree of beta-like secondary structure and the molecule cannot be described as an extended random coil. Increase of pH from 2.5 to 5.5 has no influence on the structure of the pressure-denatured state; it slightly changes the stability of the protein only. All experimental evidence indicates that the pressure-denatured states of monomeric proteins have more secondary structure than the temperature-denatured states. Different modes of denaturation, including pressure, may correlate differently with the roughness of the energy scale and slope of the folding funnel. For these reasons we have also carried out pressure-jump kinetic studies of the secondary structural evolution in the unfolding/refolding reaction of RNase A. In agreement with the theoretical model presented by Hummer et al. [(1998) Proc. Natl. Acad. Sci. U.S.A. 95, 1552-1555], the experimental data show that pressure slows down folding and unfolding kinetics (here 1-2 orders of magnitude), corresponding to an increasingly rough landscape. The kinetics remains non-two-state under pressure. Assuming a two-step folding scenario, the calculated relaxation times for unfolding of RNase A at 20 degrees C and pH 2.5 can be estimated to be tau(1) approximately 0.7 min and tau(2) approximately 17 min. The refolding process is considerably faster (tau(1) approximately 0.3 min, tau(2) approximately 4 min). Our data show that the pressure stability and pressure-induced unfolding/refolding kinetics of monomeric proteins, such as wild-type staphylococcal nuclease (WT SNase) and RNase A, may be significantly different. The differences are largely due to the four disulfide bonds in RNase A, which stabilize adjacent structures. They probably lead to the much higher denaturation pressure compared to SNase, and this might also explain why the volume change of WT SNase upon unfolding is about twice as large.     

General dynamic properties of Abeta12-36 amyloid peptide involved in Alzheimer's disease from unfolding simulation

To study the folding/unfolding properties of a beta-amyloid peptide Abeta(12-36) of Alzheimer's disease, five molecular dynamics simulations of Abeta(12-36) in explicit water were done at 450 K starting from a structure that is stable in trifluoroethanol/water at room temperature with two alpha-helices. Due to high temperature, the initial helical structure unfolded during the simulation. The observed aspects of the unfolding were as follows. 1) One helix (helix 1) had a longer life than the other (helix 2), which correlates well with the theoretically computed Phi values. 2) Temporal prolongation of helix 1 was found before unfolding. 3) Hydrophobic cores formed frequently with rearrangement of amino-acid residues in the hydrophobic cores. The formation and rearrangement of the hydrophobic cores may be a general aspect of this peptide in the unfolded state, and the structural changes accompanied by the hydrophobic-core rearrangement may lead the peptide to the most stable structure. 4) Concerted motions (collective modes) appeared to unfold helix 1. The collective modes were similar with those observed in another simulation at 300 K. The analysis implies that the conformation moves according to the collective modes when the peptide is in the initial stage of protein unfolding and in the final stage of protein folding.