Dissecting contributions to the denaturant sensitivities of proteins

Understanding the molecular basis for protein denaturation by urea and guanidinium chloride (GdmCl) should accommodate the observation that, on a molar basis, GdmCl is generally 2-2.5-fold more effective as a protein denaturant than urea. Previous studies [Smith, J. S., and Scholtz, J. M. (1996) Biochemistry 35, 7292-7297] have suggested that the effects of GdmCl on the stability of alanine-based helical peptides can be separated into denaturant and salt effects, since adding equimolar NaCl to urea enhanced urea-induced unfolding to an extent that was close to that of Gdm. We reinvestigated this observation using an alanine-based helical peptide (alahel) that lacks side chain electrostatic contributions to stability, and compared the relative denaturant sensitivities of this peptide with that of tryptophan zipper peptides (trpzip) whose native conformations are stabilized largely by cross-strand indole ring interactions. In contrast to the observations of Smith and Scholtz, GdmCl was only slightly more powerful as a denaturant of alahel than urea in salt-free buffer (the denaturant m value m(GdmCl)/m(urea) ratio = 1.4), and the denaturation of alahel by urea exhibited only a small dependence on NaCl or KCl. The trpzip peptides were much more sensitive to GdmCl than to urea (m(GdmCl)/m(urea) = 3.5-4). These observations indicate that the m(GdmCl)/m(urea) ratio of 2-2.5 for proteins results from a combination of effects on the multiple contributions to protein stability, for which GdmCl may be only slightly more effective than urea (e.g., hydrogen bonds) or considerably more effective than urea (e.g., indole-indole interactions).     

Coulomb forces control the density of the collapsed unfolded state of barstar

Although it has been recently shown that unfolded polypeptide chains undergo a collapse on transfer from denaturing to native conditions, the forces determining the dynamics and the size of the collapsed form have not yet been understood. Here, we use single-molecule fluorescence resonance energy transfer experiments on the small protein barstar to characterize the unfolded chain in guanidinium chloride (GdmCl) and urea. The unfolded protein collapses on decreasing the concentration of denaturants. Below the critical concentration of 3.5 M denaturant, the collapse in GdmCl leads to a more dense state than in urea. Since it is known that GdmCl suppresses electrostatic interactions, we infer that Coulomb forces are the dominant forces acting in the unfolded barstar under native conditions. This hypothesis is clearly buttressed by the finding of a compaction of the unfolded barstar by addition of KCl at low urea concentrations.     

Molecular basis for the effect of urea and guanidinium chloride on the dynamics of unfolded polypeptide chains

Chemical denaturants are frequently used to unfold proteins and to characterize mechanisms and transition states of protein folding reactions. The molecular basis of the effect of urea and guanidinium chloride (GdmCl) on polypeptide chains is still not well understood. Models for denaturant--protein interaction include both direct binding and indirect changes in solvent properties. Here we report studies on the effect of urea and GdmCl on the rate constants (k(c)) of end-to-end diffusion in unstructured poly(glycine-serine) chains of different length. Urea and GdmCl both lead to a linear decrease of lnk(c) with denaturant concentration, as observed for the rate constants for protein folding. This suggests that the effect of denaturants on chain dynamics significantly contributes to the denaturant-dependence of folding rate constants for small proteins. We show that this linear dependency is the result of two additive non-linear effects, namely increased solvent viscosity and denaturant binding. The contribution from denaturant binding can be quantitatively described by Schellman's weak binding model with binding constants (K) of 0.62(+/-0.01)M(-1) for GdmCl and 0.26(+/-0.01)M(-1) for urea. In our model peptides the number of binding sites and the effect of a bound denaturant molecule on chain dynamics is identical for urea and GdmCl. The results further identify the polypeptide backbone as the major denaturant binding site and give an upper limit of a few nanoseconds for residence times of denaturant molecules on the polypeptide chain.     

Amide proton exchange used to monitor the formation of a stable alpha-helix by residues 3 to 13 during folding of ribonuclease S

We make use of the known exchange rates of individual amide proton in the S-peptide moiety of ribonuclease S (RNAase S) to determine when during folding the alpha-helix formed by residues 3 to 13 becomes stable. The method is based on pulse-labeling with [3H]H2O during the folding followed by an exchange-out step after folding that removes 3H from all amide protons of the S-peptide except from residues 7 to 14, after which S-peptide is separated rapidly from S-protein by high performance liquid chromatography. The slow-folding species of unfolded RNAase S are studied. Folding takes place in strongly native conditions (pH 6.0, 10 degrees C). The seven H-bonded amide protons of the 3-13 helix become stable to exchange at a late stage in folding at the same time as the tertiary structure of RNAase S is formed, as monitored by tyrosine absorbance. At this stage in folding, the isomerization reaction that creates the major slow-folding species has not yet been reversed. Our result for the 3-13 helix is consistent with the finding of Labhardt (1984), who has studied the kinetics of folding of RNAase S at 32 degrees C by fast circular dichroism. He finds the dichroic change expected for formation of the 3-13 helix occurring when the tertiary structure is formed. Protected amide protons are found in the S-protein moiety earlier in folding. Formation or stabilization of this folding intermediate depends upon S-peptide: the intermediate is not observed when S-protein folds alone, and folding of S-protein is twice as slow in the absence of S-peptide. Although S-peptide combines with S-protein early in folding and is needed to stabilize an S-protein folding intermediate, the S-peptide helix does not itself become stable until the tertiary structure of RNAase S is formed.     

A unique and differential effect of denaturants on cofactor mediated activation of Plasmodium falciparum beta-ketoacyl-ACP reductase

The urea and guanidinium chloride (GdmCl) induced unfolding of FabG, a beta-ketoacyl-ACP reductase of Plasmodium falciparum, was examined in detail using intrinsic fluorescence of FabG, UV-circular dichroism (CD), spectrophotometric enzyme activity measurements, glutaraldehyde cross-linking, and size exclusion chromatography. The equilibrium unfolding of FabG by urea is a multistep process as compared with a two-state process by GdmCl. FabG is fully unfolded at 6.0M urea and 4.0M GdmCl. Approximately 90% of the enzyme activity could be recovered on dialyzing the denaturants, showing that denaturation by both urea and GdmCl is reversible. We found two states in the reversible unfolding process of FabG in presence of NADPH; one is an activity-enhanced state and the other, an inactive state in case of equilibrium unfolding with urea. On the contrary, in presence of NADPH, there is no stabilization of FabG in case of equilibrium unfolding with GdmCl. We hypothesize that the hydrogen-bonding network may be reorganized by the denaturant in the activity-enhanced state formed in presence of 1.0M urea, by interrupting the association between dimer-dimer interface and help in accommodating the larger substrate in the substrate binding tunnel thus, increasing the activity. Furthermore, binding of the active site organizer, NADPH leads to compaction of the FabG in presence of urea, as evident by acrylamide quenching. We have shown here for the first time, the detailed inactivation kinetics of FabG, which have not been evaluated in the past from any of the FabG family of enzymes from any of the other sources. These findings provide impetus for exploring the influences of ligands on the structure-activity relationship of Plasmodium beta-ketoacyl-ACP reductase.     

Single-molecule FRET study of denaturant induced unfolding of RNase H

Single-molecule fluorescence (F?rster) resonance energy transfer (FRET) experiments were performed on surface-immobilized RNase H molecules as a function of the concentration of the chemical denaturant guanidinium chloride (GdmCl). For comparison, we measured ensemble FRET on RNase H solutions. The single-molecule approach allowed us to study FRET distributions of the subpopulation of unfolded molecules without interference from the folded population. The unfolded ensemble experienced a continuous shift of the FRET efficiency distribution with increasing concentration of GdmCl, indicating a heterogeneous population of expanding, unfolded polypeptide chains. We have analyzed the behavior of the unfolded state quantitatively with a model in which the unfolded state is described by a continuum of substates, with the free energy of each substate linearly coupled to its m-value, the proportionality coefficient between free energy and denaturant activity. By fitting this model to the data, we have derived energetic and structural parameters that describe the unfolded state ensemble. Specifically, we have found that the average size of the unfolded state increases from 23-38 A between 0 and 6 M denaturant. Excellent agreement was achieved between the fitted model and our FRET measurements, and with previously published nuclear magnetic resonance and small-angle X-ray scattering data.     

Rapid amide proton exchange rates in peptides and proteins measured by solvent quenching and two-dimensional NMR.

In an effort to develop a more versatile quenched hydrogen exchange method for studies of peptide conformation and protein-ligand interactions, the mechanism of amide proton exchange for model peptides in DMSO-D2O mixtures was investigated by NMR methods. As in water, H-D exchange rates in the presence of 90% or 95% DMSO exhibit characteristic acid- and base-catalyzed processes and negligible water catalysis. However, the base-catalyzed rate is suppressed by as much as four orders of magnitude in 95% DMSO. As a result, the pH at which the exchange rate goes through a minimum is shifted up by about two pH units and the minimum exchange rate is approximately 100-fold reduced relative to that in D2O. The solvent-dependent decrease in base-catalyzed exchange rates can be attributed primarily to a large increase in pKa values for the NH group, whereas solvent effects on pKW seem less important. Addition of toluene and cyclohexane resulted in improved proton NMR chemical shift dispersion. The dramatic reduction in exchange rates observed in the solvent mixture at optimal pH makes it possible to apply 2D NMR for NH exchange measurements on peptides under conditions where rates are too rapid for direct NMR analysis. To test this solvent-quenching method, melittin was exchanged in D2O (pH 3.2, 12 degrees C), aliquots were quenched by rapid freezing, lyophilized, and dissolved in quenching buffer (70% DMSO, 25% toluene, 4% D2O, 1% cyclohexane, 75 mM dichloroacetic acid) for NMR analysis. Exchange rates for 21 amide protons were measured by recording 2D NMR spectra on a series of samples quenched at different times. The results are consistent with a monomeric unfolded conformation of melittin at acidic pH. The ability to trap labile protons by solvent quenching makes it possible to extend amide protection studies to peptide ligands or labile protons on the surface of a protein involved in macromolecular interactions.     

Multiple unfolding states of glutathione transferase from Physa acuta (Gastropoda [correction of Gastropada]: Physidae)

The equilibrium unfolding of the major Physa acuta glutathione transferase isoenzyme (P. acuta GST(3)) has been performed using guanidinium chloride (GdmCl), urea, and acid denaturation to investigate the unfolding intermediates. Protein transitions were monitored by intrinsic fluorescence. The results indicate that unfolding of P. acuta GST(3) using GdmCl (0-3.0M) is a multistep process, i.e., three intermediates coexist in equilibrium. The first intermediate, a partially dissociated dimer, exists at low GdmCl concentration (approximately at 0.7M). At 1.2M GdmCl, a dimeric intermediate with a compact structure was observed. This intermediate undergoes dissociation into structural monomers at 1.75M of GdmCl. The monomeric intermediate started to be completely unfolding at higher GdmCl concentrations (>1.8M). Unfolding using urea (0-7.0M) and acid-induced structures as well as the fluorescence of 8-anilino-1-naphthalenesulfonate in the presence of different GdmCl concentrations confirmed that the unfolding is a multistep process. At concentrations of GdmCl or urea less than the midpoints or at the midpoint pH (pH 4.2-4.6), the unfolding transition is protein concentration independent and involved a change in the subunit tertiary structure yielding a partially active dimeric intermediate. The binding of glutathione to the enzyme active site stabilizes the native dimeric state.     

pH dependence of hydrogen exchange from backbone peptide amides in apamin

The kinetics of hydrogen exchange of the 11 most protected backbone amides of bee venom apamin have been measured between pH 1 and pH 8.5 by using time-resolved and saturation-transfer NMR spectroscopy. The five amides most protected from base-catalyzed exchange, those of residues 5 and 12-15, show highly correlated exchange behavior in the base-catalyzed regime. It is proposed that the intramolecular hydrogen bonds stabilizing these amides define a stable cooperative unit of secondary structure in apamin (a C-terminal helix and an N-terminal beta-turn). This conformational unit is further stabilized (by 5-6 kJ mol-1) on titration of the Glu-7 side-chain carboxyl group. The relative contributions of specific intramolecular interactions to this conformational stabilization are estimated. The pHminima in the pH-dependent single amide exchange curves are compared with values predicted by correcting for sequence-dependent contributions to amide exchange rates [Molday, R. S., Englander, S. W., & Kallen, R. G. (1972) Biochemistry 11, 150-158]. The lack of correlation suggests that the "open" conformers from which amide exchange occurs are nonrandom. This conclusion is dependent on the assumption that acid-catalyzed exchange occurs via N-protonation so that residual conformational effects on exchange rates in the open conformers will affect acid- and base-catalyzed rates in approximately equal and opposite ways. A strong correlation between the measured pHminima and the amide proton chemical shifts is observed, however, and this may be most easily accommodated if acid-catalyzed exchange occurs by the imidic acid mechanism (via amide O-protonation).     

Multiple unfolded states of glutathione transferase bbGSTP1-1 by guanidinium chloride.

Inactivation, dissociation, and unfolding of the homodimeric glutathione transferase (bbGSTP1-1) from Bufo bufo embryos were investigated at equilibrium, using guanidinium chloride (GdmCl) as denaturant. Protein transitions were monitored by enzyme activity, intrinsic fluorescence, far UV circular dichroism, glutaraldehyde cross-linking, and gel-filtration chromatography. At low denaturant concentrations (less than 0.5 M), reversible inactivation of the enzyme occurs. At denaturant concentrations between 0.5 and 1.5 M the enzyme progressively dissociates into structured monomers. At higher denaturant concentrations the monomers unfold completely. Refolding studies indicate that a total reactivation occurs only by starting from the enzyme denatured at concentrations below 0.5 M. The enzyme denatured at GdmCl concentrations higher than 0.5 M only partially refolds. Globally our results indicate that unfolding of the amphibian bbGSTP1-1 is a multistep process, i.e., inactivation of the structured dimer, dissociation into partially structured monomers, followed by complete unfolding.     

Multiple unfolded states of alcohol dehydrogenase I from Kluyveromyces lactis by guanidinium chloride

Inactivation, dissociation, and unfolding of tetrameric alcohol dehydrogenase I from Kluyveromyces lactis (KlADH I) were investigated using guanidinium chloride (GdmCl) as denaturant. Protein transitions were monitored by enzyme activity, intrinsic fluorescence and gel filtration chromatography. At low denaturant concentrations (less than 0.3 M), reversible transformation of enzyme into tetrameric inactive form occurs. At denaturant concentrations between 0.3 and 0.5 M, the enzyme progressively dissociates into structured monomers through an irreversible reaction. At higher denaturant concentrations, the monomers unfold completely. Refolding studies indicate that a total reactivation occurs only with the enzyme denatured between 0 and 0.3 M GdmCl concentrations. The enzyme denatured at GdmCl concentrations higher than 0.3 M refolds only partially. All together, our results indicate that unfolding of the KlADH I is a multistep process, i.e., inactivation of the structured tetramer, dissociation into partially structured monomers, followed by complete unfolding.     

Anion modulation of the 1H/2H exchange rates in backbone amide protons monitored by NMR spectroscopy

The ability of three anionic cosolutes (sulfate, thiocyanate, and chloride) in modulating the (1)H/(2)H exchange rates for backbone amide protons has been investigated using nuclear magnetic resonance (NMR) for two different proteins: the IGg-binding domain of protein L (ProtL) and the glucose-galactose-binding protein (GGBP). Our results show that moderate anion concentrations (0.2 M-1 M) regulate the exchange rate following the Hofmeister series: Addition of thiocyanate increases the exchange rates for both proteins, while sulfate and chloride (to a less extent) slow down the exchange reaction. In the presence of the salt, no alteration of the protein structure and minimal variations in the number of measurable peaks are observed. Experiments with model compounds revealed that the unfolded state is modulated in an equivalent way by these cosolutes. For ProtL, the estimated values for the local free energy change upon salt addition (m (3,DeltaG )) are consistent with the previously reported free energy contribution from the cosolute's preferential interaction/exclusion term indicating that nonspecific weak interactions between the anion and the amide groups constitute the dominant mechanism for the exchange-rate modulation. The same trend is also found for GGBP in the presence of thiocyanate, underlining the generality of the exchange-rate modulation mechanism, complementary to more investigated effects like the electrostatic interactions or specific anion binding to protein sites.     

Anion shielding of electrostatic repulsions in transthyretin modulates stability and amyloidosis: insight into the chaotrope unfolding dichotomy

The balance between stabilizing forces and the localized electrostatic repulsions destabilizing the transthyretin (TTR) tetramer is tunable via anion shielding. The two symmetrical anion interaction sites in TTR are comprised of residues Lys15 and Lys15' from opposing subunits on the periphery of the two thyroxine binding sites. These epsilon-ammonium groups repel one another and destabilize the tetramer, unless an appropriate anion is present, which stabilizes the tetramer. Chaotrope denaturation of TTR exhibits unusual behavior in that urea appears to be a stronger denaturant than GdmCl (guanidinium chloride), even though GdmCl is typically twice as powerful as a denaturant. The shift in the midpoint of the urea denaturation curve to higher concentrations as well as the increase in the mole fraction of tetramer that is highly resistant to denaturation with increasing KCl concentration provides strong evidence that anion shielding stabilizes the TTR tetramer. A consequence of tetramer stabilization is folding hysteresis, because the high GdmCl concentrations required to denature the anion-stabilized tetramer do not allow refolding of the unfolded monomers. The formation of amyloid fibrils by TTR requires that its normal tetrameric structure dissociate to alternatively folded monomers, a process mediated by acidification (pH 5-4). This process is inhibited by Cl(-) ions in a concentration-dependent fashion. Chloride ion may not be the relevant physiological TTR stability modulator, but it is the main focus of these studies explaining the hysteresis observed in the denaturation and refolding studies with GdmCl.     

Denaturation of phosphofructokinase-1 from Saccharomyces cerevisiae by guanidinium chloride and reconstitution of the unfolded subunits to their catalytically active form

Unfolding and refolding of heterooctameric phosphofructokinase-1 from Saccharomyces cerevisiae were investigated by application of kinetic, hydrodynamic, and spectroscopic methods and by use of guanidinium chloride (GdmCl) as denaturant. Inactivation of the enzyme starts at about 0.3 M GdmCl and undergoes a sharp unfolding transition in a narrow range of the denaturant concentration. The inactivation is accompanied by a dissociation of the enzyme into dimers (at 0.6 M GdmCl), which could be detected by changes of the circular dichroism and intrinsic fluorescence. Protein aggregates were observed from 0.7 to 1.5 M GdmCl that unfold at higher denaturant concentrations. Refolding of chemically denatured phosphofructokinase proceeds as a stepwise process via the generation of elements of secondary structure, the formation of assembly-competent monomers that associate to heterodimers and the assembly of dimers to heterotetramers and heterooctamers. The assembly reactions seem to be rate-limiting. Recovery of the enzyme activity (maximum 65%) competes with an nonproductive aggregation of the subunits. alpha-Cyclodextrin functions as an artificial chaperone by preventing aggregation of the subunits, whereas ATP is suggested to support the generation of heterodimers that are competent to a further assembly.     

Nonrandom structure in the urea-unfolded Escherichia coli outer membrane protein X (OmpX)

On the basis of sequence-specific resonance assignments for the complete polypeptide backbone and most of the amino acid side chains by heteronuclear nuclear magnetic resonance (NMR) spectroscopy, the urea-unfolded form of the outer membrane protein X (OmpX) from Escherichia coli has been structurally characterized. (1)H-(1)H nuclear Overhauser effects (NOEs), dispersion of the chemical shifts, amide proton chemical shift temperature coefficients, amide proton exchange rates, and (15)N[(1)H]-NOEs show that OmpX in 8 M urea at pH 6.5 is globally unfolded, but adopts local nonrandom conformations in the polypeptide segments of residues 73-82 and 137-145. For these two regions, numerous medium-range and longer-range NOEs were observed, which were used as the input for structure calculations of these polypeptide segments with the program DYANA. The segment 73-82 forms a quite regular helical structure, with only loosely constrained amino acid side chains. In the segment 137-145, the tryptophan residue 140 forms the core of a small hydrophobic cluster. Both nonrandom structures are present with an abundance of about 25% of the protein molecules. The sequence-specific NMR assignment and the physicochemical characterization of urea-denatured OmpX presented in this paper are currently used as a platform for investigations of the folding mechanism of this integral membrane protein.     

Folding and association of an extremely stable dimeric protein from Sulfolobus islandicus

ORF56 is a plasmid-encoded protein from Sulfolobus islandicus, which probably controls the copy number of the pRN1 plasmid by binding to its own promotor. The protein showed an extremely high stability in denaturant, heat, and pH-induced unfolding transitions, which can be well described by a two-state reaction between native dimers and unfolded monomers. The homodimeric character of native ORF56 was confirmed by analytical ultracentrifugation. Far-UV circular dichroism and fluorescence spectroscopy gave superimposable denaturant-induced unfolding transitions and the midpoints of both heat as well as denaturant-induced unfolding depend on the protein concentration supporting the two-state model. This model was confirmed by GdmSCN-induced unfolding monitored by heteronuclear 2D NMR spectroscopy. Chemical denaturation was accomplished by GdmCl and GdmSCN, revealing a Gibbs free energy of stabilization of -85.1 kJ/mol at 25 degrees C. Thermal unfolding was possible only above 1 M GdmCl, which shifted the melting temperature (t(m)) below the boiling point of water. Linear extrapolation of t(m) to 0 M GdmCl yielded a t(m) of 107.5 degrees C (5 microM monomer concentration). Additionally, ORF56 remains natively structured over a remarkable pH range from pH 2 to pH 12. Folding kinetics were followed by far-UV CD and fluorescence after either stopped-flow or manual mixing. All kinetic traces showed only a single phase and the two probes revealed coincident folding rates (k(f), k(u)), indicating the absence of intermediates. Apparent first-order refolding rates depend linearly on the protein concentration, whereas the unfolding rates do not. Both lnk(f) and lnk(u) depend linearly on the GdmCl concentration. Together, folding and association of homodimeric ORF56 are concurrent events. In the absence of denaturant ORF56 refolds fast (7.0 x 10(7)M(-1)s(-1)) and unfolds extremely slowly (5.7 year(-1)). Therefore, high stability is coupled to a slow unfolding rate, which is often observed for proteins of extremophilic organisms.     

IR spectra of cytochrome c denatured with deuterated guanidine hydrochloride show increase in beta sheet

Attenuated total reflectance Fourier transform IR (ATR-FTIR) spectra are obtained for horse heart ferricytochrome c in solutions of 0-7M guanidine hydrochloride and deuterated guanidine hydrochloride. Substitutions of deuterium for hydrogen in both the denaturant and protein provide resolvable amide I spectra over a wide range of denaturant concentrations. Deuteration enhances the ability to measure the true protein IR spectrum in the amide I region in which the secondary structure can be deduced, because spectra in D(2)O are less prone to spectral distortion upon background denaturant subtraction than spectra in H(2)O. Other investigators studying equilibrium unfolded cytochrome c were limited to guanidine concentrations below 3.0M because of detector saturation. Detector saturation is avoided with the use of ATR-FTIR spectroscopy, allowing one to obtain protein spectra at high denaturant concentrations. Second derivative spectra of samples show reductions in alpha helix and increases in beta sheet at high denaturant concentrations, contrary to expectations of finding primarily a random coil secondary structure. Using this new technique, the protein was estimated to consist of 51% beta sheet and only 15% random coil in the presence of 6.6M deuterated guanidine hydrochloride.     

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.     

Hydrogen exchange from identified regions of the S-protein component of ribonuclease as a function of temperature,pH, and the binding of S-peptide

A medium resolution hydrogen exchange method (Rosa & Richards, 1979) has been used to measure the average rates of amide hydrogen exchange for known segments of the S-protein portion of ribonuclease-S. The analytical procedure permitted exchange rates to be monitored for seven S-protein fragments distributed throughout the structure, including regions of α-helix and β-sheet. Kinetics were measured as a function of pH, temperature and S-peptide binding.The pH dependence of exchange from isolated S-protein between pH 2·8 and pH 7·0 was found to deviate significantly from a first-order dependence on hydroxide ion concentration. The protection against exchange with increasing pH appeared to be closely related to the electrostatic stabilization of S-protein. It is suggested that such favorable electrostatic interactions result in increased energy barriers to the conformational fluctuations that provide solvent access to the time-average crystallographic structure. This explanation of the observed correlation between stability and exchange kinetics is also consistent with the calculated apparent activation energies for exchange from S-protein between 5·5 and 20 °C.S-peptide binding dramatically slows exchange from many S-protein sites, even those distant from the area of S-peptide contact. Interestingly, the effects of complex formation are not evenly propagated throughout S-protein. The most significantly perturbed sites (≥10³-fold reduction in exchange rate constants) lie within fragments derived from regions of secondary structure. Exchange from several other fragments is not significantly affected. The S-peptide—S-protein dissociation constant at neutral pH is so small that the measured exchange must have occurred from the complex and not from the dissociated parts.     

Protection of amide protons in folding intermediates of ribonuclease A measured by pH-pulse exchange curves

pH-pulse exchange curves have been measured for samples taken during the folding of ribonuclease A. The curve gives the number of protected amide protons remaining after a 10-s pulse of exchange at pHs from 6.0 to 9.5, at 10 degrees C. Amide proton exchange is base catalyzed, and the rate of exchange increases 3000-fold between pH 6.0 and pH 9.5. The pH at which exchange occurs depends on the degree of protection against exchange provided by structure. Pulse exchange curves have been measured for samples taken at three times during folding, and these are compared to the pulse exchange curves of N, the native protein, of U, the unfolded protein in 4 M guanidinium chloride, and of IN, the native-like intermediate obtained by the prefolding method of Schmid. The results are used to determine whether folding intermediates are present that can be distinguished from N and U and to measure the average degree of protection of the protected protons in folding intermediates. The amide (peptide NH) protons of unfolded ribonuclease A were prelabeled with 3H by a previous procedure that labels only the slow-folding species. Folding was initiated at pH 4.0, 10 degrees C, where amide proton exchange is slower than the folding of the slow-folding species. Samples were taken at 0-, 10-, and 20-s folding, and their pH-pulse exchange curves were measured.(ABSTRACT TRUNCATED AT 250 WORDS)