Initial protein concentration and residual denaturant concentration strongly affect the batch refolding of hen egg white lysozyme

The effects of several variables on the refolding of hen egg white lysozyme have been studied. Lysozyme was denatured in both urea, and guanidine hydrochloride (GuHCl), and batch refolded by dilution (100 to 1000 fold) into 0.1M Tris-HCl, pH 8.2, 1 mM EDTA, 3 mM reduced glutathione and 0.3 mM oxidised glutathione. Refolding was found to be sensitive to temperature, with the highest refolding yield obtained at 50°C. The apparent activation energy for lysozyme refolding was found to be 56 kJ/mol. Refolding by dilution results in low concentrations of both denaturant and reducing agent species. It was found that the residual concentrations obtained during dilution (100-fold dilution: [GuHCl]=0.06 mM, [DTT]=0.15 mM) were significant and could inhibit lysozyme refolding. This study has also shown that the initial protein concentration (1–10 mg/mL) that is refolded is an important parameter. In the presence of residual GuHCl and DTT, higher refolding yields were obtained when starting from higher initial lysozyme concentrations. This trend was reversed when residual denaturant components were removed from the refolding buffer.     

Does beta-lactoglobulin denaturation occur via an intermediate state?

The denaturation of beta-lactoglobulin (BLG) in the presence of urea and GuHCl has been investigated at different pH values with various spectroscopic techniques. The equilibrium denaturation free energy values, obtained by linearly extrapolating the data to vanishing denaturant (DeltaG(D)(H2O)), are compared and discussed. The fit of the spectroscopic data monitoring the denaturation of BLG has been approached, at first, with a two-state model that describes the protein transition from the folded state (at each pH and in the absence of denaturant) to the denatured state, but in particular, along the GuHCl denaturation pathway some evidence is found of the presence of an intermediate state. Time-resolved fluorescence experiments performed on the BLG-ANS (1-anilino-8-naphthalenesulfonate) complex help to understand the results. Fluorescence polarization anisotropy (FPA) measurements accompanying the denaturation process show the presence of a fast rotational diffusion of the ANS probe, and the data are interpreted in terms of local fluctuations of a still structured tract of the denatured protein where the probe is bound.     

Thermodynamic investigations of proteins. II. Calorimetric study of lysozyme denaturation by guanidine hydrochloride.

The thermodynamic parameters of the denaturation of lysozyme are determined at various temperatures (25-60 degrees C) by isothermal calorimetric titrations with guanidine hydrochloride (GuHCl) and by scanning calorimetry in the presence of GuHCl. An approach for the determination of the enthalpy of preferential binding of GuHCl is proposed. It has been shown from GuHCl denaturation experiments that the net enthalpies of denaturation and the denaturational change in the heat capacity of protein can be obtained if preferential binding is taken into consideration. These results are nearly the same as in the case of thermal denaturation in the absence of denaturants. It is concluded that the states of both heat- and GuHCl-denatured lysozyme are thermodynamically indistinguishable.     

Optical spectroscopic differentiation of various equilibrium denatured states of horse cytochrome c

Thermal unfolding of cytochrome c (cyt c) from several states has been studied using equilibrium spectroscopic techniques. CD in the uv, vibrational circular dichroism, infrared, and uv-vis absorption spectra measured at various temperatures, pHs, salt concentrations, and GuHCl concentrations are used to show the conformational as well as heme structural differences between native and various denatured states. The difference in thermal denaturation behaviors of cyt c starting from acid denatured, molten globule (MG), and the A and native states are explored. Different final high temperature states were observed for cytochrome c unfolding from four different initial states (native, MG, A, and acid denatured state) by electronic CD, Fourier transform infrared (FTIR), and vibrational CD (VCD). Consistent with this, different thermal unfolding pathways for the MG and A states are suggested by the FTIR and VCD data for this process.     

Refolding of partially and fully denatured lysozymes

Lysozyme refolding with high yields sometimes results from incomplete denaturation. Dithiothreitol (DTT) is a reductant commonly used to reduce and unfold disulfide-stabilized lysozymes. Through the use of fluorescence spectroscopy to access the extent of denaturation, we found that the rate and extent of denaturation highly depended on the concentration of DTT. Further, the denaturation exhibited a two-phase transition at a high DTT concentration with DTT at >100 mM and long denaturation time (>24 h) being needed for complete denaturation. A low DTT concentration and a short denaturation time resulted in fast refolding with high activity recovery, while a high DTT concentration and a long denaturation time resulted in slow refolding with low activity recovery. Hence, the renaturation of disulfide-containing lysozyme was highly affected by the extent of denaturation.     

Reductive cleavage and regeneration of the disulfide bonds inStreptomyces subtilisin inhibitor (SSI) as studied by the carbonyl13C NMR resonances of cysteinyl residues

Summary Four enhanced carbonyl carbon resonances were observed whenStreptomyces subtilisin inhibitor (SSI) was labeled by incorporating specifically labeled [1-¹³C]Cys. The¹³C signals were assigned by the¹⁵N,¹³C double-labeling method along with site-specific mutagenesis. Changes in the spectrum of the labeled protein ([C]SSI) were induced by reducing the disulfide bonds with various amounts of dithiothreitol (DTT). The results indicate that, in the absence of denaturant, the Cys⁷¹-Cys¹⁰¹ disulfide bond of each SSI subunit can be reduced selectively. This disulfide bond, which is in the vicinity of the reactive site scissile bond Met⁷³-Val⁷⁴, is more accessible to solvent than the other disulfide bond. Cys³⁵-Cys⁵⁰, which is embedded in the interior of SSI. This half-reduced SSI had 65% of the inhibitory activity of native SSI and maintained a conformation similar to that of the fully oxidized SSI. Reoxidation of the half reduced-folded SSI by air regenerates fully active SSI which is indistinguishable with intact SSI by NMR. In the presence of 3 M guanidine hydrochloride (GuHCl), however, both disulfide bonds of each SSI subunit were readily reduced by DTT. The fully reduced-unfolded SSI spontaneously refolded into a native-like structure (fully reduced-folded state), as evidenced by the Cys carbonyl carbon chemical shifts, upon removing GuHCl and DTT from the reaction mixture. The time course of disulfide bond regeneration from this state by air oxidation was monitored by following the NMR spectral changes and the results indicated that the disulfide bond between Cys⁷¹ and Cys¹⁰¹ regenerates at a much faster rate than that between Cys³⁵ and Cys⁵⁰.Nomenclature of the various states of SSI that are observed in the present study Fully oxidized-folded native or intact (without GuHCl or DTT) - half reduced-folded (Cys⁷¹-Cys¹⁰¹ reduced; DTT without GuHCl) - inversely half reduced-folded (Cys³⁵-Cys⁵⁰ reduced; a reoxidation intermediate from fully reduced-folded state) - fully reduced-unfolded (reduced by DTT in the presence of GuHCl) - fully reduced-folded (an intermediate state obtained by removing DTT and GuHCl from the fully reduced-unfolded SSI reaction mixture)     

Denaturation and proteolytic digestion of porcine low-density lipoprotein in aqueous guanidine hydrochloride solutions.

The denaturation of porcine low-density lipoprotein (LDL) in aqueous guanidine hydrochloride (GuHCl) was studied by flotation velocity experiments, optical rotatory dispersion and fluorescence spectroscopy. The denaturation of LDL occurred between 2 and 4M GuGCl, where small sigmoidal changes in iptical rotation and fluorescence intensity were noted. The hydrated density of the native LDL was 1.036g/cm-3 and this remained constant upon denaturation in 4M GuHCl. The slope of the flotation coefficient-solvent density curve was 35 per cent less for denatured LDL than for the native LDL. Since there is no indication of splitting of LDL in 4M GuHCl, it is natural to interpret the result in terms of an increase of the translational frictional coefficient by 50 per cent. The observed changes in optical rotation, fluorescence intensity and flotation coefficient in 4M GuHCl were readily reversed and native LDL was recovered after removal of GuHCl by dialysis. Proteolytic treatment of denatured LDL produced digested LDL which had a hydrated density of 1.021g/cm-3 corresponding to the loss of 30 per cent of apo-LDL. The digested LDL behaved like a compact, globular particle in aqueous NaCl solution and in 4M GuHCl. These results can best be interpreted by a model of the LDL particle in which approximately 30 per cent of apo-LDL is exposed to the solvent, such that it can be reversibly denatured by GuHCl and at the same time is easily avalable to proteolytic enzymes, whereas the rest of apo-LDL is tightly associated with lipids and possibly buried inside the lipid moiety. SDS-polyacrylamide gel electrophoresis of the digested LDL revealed four major peptide fragments with sizes ranging from 70,000 to 100,000 daltons. We believe that the method and results described in this paper will have meaningful applications in the study of membrane proteins.     

Folding studies of the cysteine proteinase inhibitor--human stefin A

Reversible GuHCl denaturation of human stefin A (25 degrees C, pH 8) was monitored by the tyrosine fluorescence, by circular dichroism in the near UV and by circular dichroism in the far UV. In each case a midpoint of 2.8 +/- 0.1 M GuHCl was obtained, demonstrating the cooperativity of the denaturation. Kinetics of the slow folding on diluting the protein from the GuHCl denatured state, was also measured by the three spectroscopic probes (10 degrees C, pH 8). Results conform to a sequential mechanism. Denaturant concentration and temperature dependence of the slow folding were measured by fluorescence. From a linear Arrhenius plot the Ea of 100 +/- 5 kJ/mol was read. 'Double mixing' experiments revealed a slow reaction going on in the unfolded state which influenced the amplitude of the fluorescence changes. 'Double mixing' experiments performed by FPLC have shown that the folding itself, i.e., the formation of a compact state, was not dependent on the time spent under unfolding conditions.     

Synchrotron SAXS studies on the structural stability of Carcinus aestuarii hemocyanin in solution

The effect of GuHCl and of NaCl on the structural properties of the hemocyanin (Hc) from Carcinus aestuarii has been studied by small angle x-ray scattering (SAXS) using synchrotron radiation. SAXS data collected as a function of perturbant concentration have been used to analyze conformational states of hexameric holo and apoHc as well as the holo and apoforms of the monomeric subunit CaeSS2. In the case of the holoprotein in GuHCl, two concentration domains were identified: at lower concentration, the perturbant induces aggregation of Hc molecules, whereas at higher concentration the aggregates dissociate with concomitant denaturation of the protein. In contrast, with apoHc the denaturation occurs at rather low GuHCl, pointing to an important effect of the active site bound copper for the stabilization of Hc tertiary structure. The effects of NaCl are similar to those of GuHCl as far as CaeSS2 is concerned, namely oligomerization precedes denaturation, whereas in the case of the hexameric form no aggregation occurs. To improve data analysis, on the basis of the current models for Hc monomers and oligomers, the fraction of each aggregation state and/or unfolded protein has been determined by fitting experimental SAXS curves with form factors calculated from Monte Carlo methods. In addition, a global analysis has been carried out on the basis of a thermodynamic model involving an equilibrium between a monomer in a nativelike and denatured form as well as a class of equilibria among the monomer and other aggregates.     

Unusually slow denaturation and refolding processes of pyrrolidone carboxyl peptidase from a hyperthermophile are highly cooperative: real-time NMR studies

The refolding rate of heat-denatured cysteine-free pyrrolidone carboxyl peptidase (PCP-0SH) from Pyrococcus furiosus has been reported to be unusually slow under some conditions. To elucidate the structural basis of the unusually slow kinetics of the protein, the denaturation and refolding processes of the PCP-0SH were investigated using a real-time 2D (1)H-(15)N HSQC and CD experiments. At 2 M urea denaturation of the PCP-0SH in the acidic region, all of the native peaks in the 2D HSQC spectrum completely disappeared. The conformation of the PCP-0SH just after removal of 6 M GuHCl could be observed as a stable intermediate (D(1) state) in 2D HSQC and CD experiments, which is similar to a molten globule structure. The D(1) state of the PCP-0SH, which is the initial state of refolding, corresponded to the state at 2 M urea and seemed to be the denatured state in equilibrium with the native state under the physiological conditions. The refolding of PCP-0SH from the D(1) state to the native state could be observed to be highly cooperative without any intermediates between them, even if the refolding rate was quite slow. In the higher concentration of denaturants, PCP-0SH showed HSQC and CD spectra characteristic of completely unfolded proteins called the D(2) state. The unusually slow refolding rate was discussed as originating in the conformations in the transition state and/or the retardation of reorganization in an ensemble of nonrandom denatured structures in the D(1) state.     

The process of amyloid-like fibril formation by methionine aminopeptidase from a hyperthermophile, Pyrococcus furiosus

Amyloid is associated with serious diseases including Alzheimer's disease and senile-systemic amyloidosis due to misfolded proteins. In the course of study of the denaturation process of methionine aminopeptidase (MAP) from the hyperthermophile P. furiosus, we found that MAP forms amyloid-like fibrils, and we then investigated the mechanism of amyloid fibril formation. The kinetic experiments on denaturation monitored by CD at 222 nm indicated that MAP in the presence of 3.37 M GuHCl at pH 3.31 changed to a conformation containing a considerable content of beta-sheet structure after the destruction of the alpha-helical structure. MAP in this beta-rich conformation was highly associated, and its stability was remarkably high: the midpoint of the GuHCl denaturation curve was 4.82 M at pH 3.0, and a thermal transition was not observed up to 125 degrees C by calorimetry. The amyloid-like fibril formation of MAP was confirmed by Congo red staining with a typical peak at 542 nm in the difference spectrum, showing a cross-beta X-ray diffraction pattern with a clear sharp reflection at 4.7 A and a characteristic unbranched fibrillar appearance with a length of about 1000 A and a diameter of about 70 A in the electron micrographs. Present results indicate that the amyloid-like form of MAP appears just after the protein is almost completely denatured, and even highly stable proteins can also form amyloid-like conformation under conditions where the denatured state of the protein is abundantly populated.     

Role of amino acid residues at turns in the conformational stability and folding of human lysozyme

To clarify the role of amino acid residues at turns in the conformational stability and folding of a globular protein, six mutant human lysozymes deleted or substituted at turn structures were investigated by calorimetry, GuHCl denaturation experiments, and X-ray crystal analysis. The thermodynamic properties of the mutant and wild-type human lysozymes were compared and discussed on the basis of their three-dimensional structures. For the deletion mutants, Delta47-48 and Delta101, the deleted residues are in turns on the surface and are absent in human alpha-lactalbumin, which is homologous to human lysozyme in amino acid sequence and tertiary structure. The stability of both mutants would be expected to increase due to a decrease in conformational entropy in the denatured state; however, both proteins were destabilized. The destabilizations were mainly caused by the disappearance of intramolecular hydrogen bonds. Each part deleted was recovered by the turn region like the alpha-lactalbumin structure, but there were differences in the main-chain conformation of the turn between each deletion mutant and alpha-lactalbumin even if the loop length was the same. For the point mutants, R50G, Q58G, H78G, and G37Q, the main-chain conformations of these substitution residues located in turns adopt a left-handed helical region in the wild-type structure. It is thought that the left-handed non-Gly residue has unfavorable conformational energy compared to the left-handed Gly residue. Q58G was stabilized, but the others had little effect on the stability. The structural analysis revealed that the turns could rearrange the main-chain conformation to accommodate the left-handed non-Gly residues. The present results indicate that turn structures are able to change their main-chain conformations, depending upon the side-chain features of amino acid residues on the turns. Furthermore, stopped-flow GuHCl denaturation experiments on the six mutants were performed. The effects of mutations on unfolding-refolding kinetics were significantly different among the mutant proteins. The deletion/substitutions in turns located in the alpha-domain of human lysozyme affected the refolding rate, indicating the contribution of turn structures to the folding of a globular protein.     

Differences in the effects of TFE on the folding pathways of human stefins A and B.

Trifluoroethanol (TFE) has been used to probe differences in the stability of the native state and in the folding pathways of the homologous cysteine protein inhibitors, human stefin A and B. After complete unfolding in 4.5 mol/L GuHCl, stefin A refolded in 11% (vol/vol) TFE, 0.75 mol/L GuHCl, at pH 6.0 and 20 degrees C, with almost identical first-order rate constants of 4.1 s-1 and 5.5 s-1 for acquisition of the CD signal at 230 and 280 nm, respectively, rates that were markedly greater than the value of 0.11 s-1 observed by the same two probes when TFE was absent. The acceleration of the rates of refolding, monitored by tyrosine fluorescence, was maximal at 10% (vol/vol) TFE. Similar rates of refolding (6.2s-1 and 7.2 s-1 for ellipticity at 230 and 280 nm, respectively) were observed for stefin A denatured in 66% (vol/vol) TFE, pH 3.3, when refolding to the same final conditions. After complete unfolding in 3.45 mol/L GuHCl, stefin B refolded in 7% (vol/vol) TFE, 0.57 mol/L GuHCl, at pH 6.0 and 20 degrees C, with a rate constant for the change in ellipticity at 280 nm of 32.8 s-1; this rate was only twice that observed when TFE was absent. As a major point of distinction from stefin A, the refolding of stefin B in the presence of TFE showed an overshoot in the ellipticity at 230 nm to a value 10% greater than that in the native protein; this signal relaxed slowly (0.01 s-1) to the final native value, with little concomitant change in the near-ultraviolet CD signal; the majority of this changes in two faster phases. After denaturation in 42% (vol/vol) TFE, pH 3.3, the kinetics of refolding to the same final conditions exhibited the same rate-limiting step (0.01 s-1) but were faster initially. The results show that similarly to stefin A, stefin B forms its hydrophobic core and predominant part of the tertiary structure faster in the presence of TFE. The results imply that the alpha-helical intermediate of stefin B is highly structured. Proteins 1999;36:205-216.     

The denatured state of N-PGK is compact and predominantly disordered

The organisation of the structure present in the chemically denatured N-terminal domain of phosphoglycerate kinase (N-PGK) has been determined by paramagnetic relaxation enhancements (PREs) to define the conformational landscape accessible to the domain. Below 2.0 M guanidine hydrochloride (GuHCl), a species of N-PGK (denoted I_b) is detected, distinct from those previously characterised by kinetic experiments [folded (F), kinetic intermediate (I_k) and denatured (D)]. The transition to I_b is never completed at equilibrium, because F predominates below 1.0 M GuHCl. Therefore, the ability of PREs to report on transient or low population species has been exploited to characterise I_b. Five single cysteine variants of N-PGK were labelled with the nitroxide electron spin-label MTSL [(1-oxyl-2,2,5,5-tetramethyl-3-pyrroline-3-methyl)methanesulfonate] and the denaturant dependences of the relaxation properties of the amide NMR signals between 1.2 and 3.6 M GuHCl were determined. Significant PREs for I_b were obtained, but these were distributed almost uniformly throughout the sequence. Furthermore, the PREs indicate that no specific short tertiary contacts persist. The data indicate a collapsed state with no coherent three-dimensional structure, but with a restricted radius beyond which the protein chain rarely reaches. The NMR characteristics of I_b indicate that it forms from the fully denatured state within 100 μs, and therefore a rapid collapse is the initial stage of folding of N-PGK from its chemically denatured state. By extrapolation, I_b is the predominant form of the denatured state under native conditions, and the non-specifically collapsed structure implies that many non-native contacts and chain reversals form early in protein folding and must be broken prior to attaining the native state topology.     

Temperature-induced dissociation of protein aggregates: accessing the denatured state

The thermal denaturation of lysozyme and ribonuclease A (RNase A) under reducing and nonreducing conditions at neutral pH has been monitored by Fourier transform infrared spectroscopy. In the absence of the reductant, lysozyme and RNase A undergo apparent three- and two-state denaturation, respectively, as observed from the conformation-sensitive amide I' band. For both proteins the hydrogen-deuterium exchange takes place at lower temperatures than the main denaturation temperatures, suggesting that a transient denaturation mechanism occurs. The observed transition at 51.2 degrees C during the denaturation of lysozyme is attributed to this transient effect, rather than to the loss of tertiary structure. Under reducing conditions lysozyme aggregates during the heating phase, whereas RNase A shows only a minor aggregation, which further increases during the cooling step. The reduced stability of both proteins can be correlated with the transient denaturation behavior, which is also suggested to be involved in protein aggregation at physiologically relevant temperatures. In addition, it is shown that when the temperature is further increased, the amorphous aggregates dissociate. Comparison of the dissociated states with the denatured states obtained under nonreducing conditions indicates that these states have the same conformation. By using a two-dimensional correlation analysis we were able to show that the dissociation is preceded by a conformational change. It is argued that this extends to other types of perturbation.     

Kietics of thermal unfolding and refolding of thermostable phosphoglycerate kinase.

The kinetics of denaturation by guanidine hydrochloride (GuHCl) of a thermostable phosphoglycerate kinase (PGK) extracted from Thermus thermophilus and of yeast PGK at neutral pH were studied by circular dichroism. Denaturation by GuHCl proceeded as a first-order reaction. The activation free energy of the denaturation reactions (delta Gf not identical to ) in the absence of GuHCl was estimated to be 32.7 kcal/mol for T. thermophilus PGK and 27.9 kcal/mol for yeast PGK (at 25 degrees C). Measurements of the rate constants at various temperatures indicated that delta Gf not identical to has maximum values at 29 degrees C for T. thermophilus PGK and at 20 degrees C for yeast PGK, and that the temperature dependences of delta Gf not identical to, delta Hf not identical to, and delta Sf not identical to for T. thermophilus PGK are smaller than those of yeast PGK. Values of delta Sf not identical to for thermal denaturation for both PGK's are approximately 200 e.u.     

Kinetic analysis of the thermal stability of the photosynthetic reaction center from Rhodobacter sphaeroides

The temperature-induced denaturation of the photosynthetic reaction center from Rhodobacter sphaeroides has been studied through the changes that occur in the absorption spectrum of the bound chromophores on heating. At elevated temperatures, the characteristic absorbance bands of the bacteriochlorins bound to the polypeptides within the reaction center are lost, and are replaced by features typical of unbound bacteriochlorophyll and bacteriopheophytin. The kinetics of the spectral changes cannot be explained by a direct conversion from the functional to the denatured form of the protein, and require the presence of at least one intermediate. Possible mechanisms for the transformation via an intermediate are examined using a global analysis of the kinetic data, and the most likely mechanism is shown to involve a reversible transformation between the native state and an off-pathway intermediate, coupled to an irreversible transformation to the denatured state. The activation energies for the transformations between the three components are calculated from the effect of temperature on the individual rate constants, and the likely structural changes of the protein during the temperature-induced transformation are discussed.     

A laser Raman study of lysozyme denaturation

The Raman spectrum of chemically denatured lysozyme was studied. The denaturants studied included dimethyl sulfoxide, LiBr, guanidine · HCl, sodium dodecyl sulfate, and urea. Previous studies have shown that the amide I and amide III regions of the Raman spectrum are sensitive to the nature of the hydrogen bond involving the amide group. The intensity of the amide III band at 1260 cm^?1 (assigned to strongly hydrogen-bonded α-helix structure) relative to the intensity of the amide III band near 1240 cm^?1 (assigned to less strongly hydrogen-bonded groups) is used as a parameter for comparison with other physical parameters used to assess denaturation. The correlation between this Raman parameter and denaturation as evidenced by enzyme activity and viscosity measurements is good, leading to the conclusion that the amide III Raman spectrum is useful for assessing the degree of denaturation. The Raman spectrum clearly depends on the type of denaturant employed, suggesting that there is not one unique denatured state for lysozyme. The data, as interpreted, place constraints on the possible models for lysozyme denaturation. One of these is that the simple two-state model does not seem consistent with the observed Raman spectral changes.     

Structural and thermodynamic characterization of T4 lysozyme mutants and the contribution of internal cavities to pressure denaturation

Using small-angle X-ray scattering (SAXS) and tryptophan fluorescence spectroscopy, we have identified multiple compact denatured states of a series of T4 lysozyme mutants that are stabilized by high pressures. Recent studies imply that the mechanism of pressure denaturation is the penetration of water into the protein rather than the transfer of hydrophobic residues into water. To investigate water penetration and the volume change associated with pressure denaturation, we studied the solution behavior of four T4 lysozyme mutants having different cavity volumes at low and neutral pH up to a pressure of 400 MPa (0.1 MPa = 0.9869 atm). At low pH, L99A T4 lysozyme expanded from a compact folded state to a partially unfolded state with a corresponding change in radius of gyration from 17 to 32 A. The volume change upon denaturation correlated well with the total cavity volume, indicating that all of the molecule's major cavities are hydrated with pressure. As a direct comparison to high-pressure crystal structures of L99A T4 lysozyme solved at neutral pH [Collins, M. D., Hummer, G., Quillin, M. L., Matthews, B. W., and Gruner, S. M. (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 16668-16671], pressure denaturation of L99A and the structurally similar L99G/E108V mutant was studied at neutral pH. The pressure-denatured state at neutral pH is even more compact than at low pH, and the small volume changes associated with denaturation suggest that the preferential filling of large cavities is responsible for the compactness of the pressure-denatured state. These results confirm that pressure denaturation is characteristically distinct from thermal or chemical denaturation.     

NMR studies on the induced denaturation of lysozyme by guanidine hydrochloride in the presence of the inhibitor (NAG)3

The stabilization of the folded conformation of lysozyme, arising from the binding of the inhibitor (NAG)3 against induced denaturation, is demonstrated from the 1H-nmr spectra of the enzyme. The nmr spectra reveal that the binding of the denaturant (GuHCl) to the enzyme is associated with changes in the conformation of the enzyme. The binding site of the inhibitor site C also serves as one of the binding sites of GuHCl. The observation that higher denaturant concentrations are required in the unfolding of Lys-(NAG)3 as compared to free Lys can be explained partly in terms of the existence of a competitive binding to the enzyme involving the (NAG)3 and GuHCl molecules.