Preferential interactions of urea with lysozyme and their linkage to protein denaturation

The interactions involved in the denaturation of lysozyme in the presence of urea were examined by thermal transition studies and measurements of preferential interactions of urea with the protein at pH 7.0, where it remains native up to 9.3 M urea, and at pH 2.0, where it undergoes a transition between 2.5 and 5.0 M urea. The destabilization of lysozyme by urea was found to follow the linear dependence on urea molar concentration, M(u), DeltaG(u)(o)=DeltaG(w)(o)-2.1 M(u), over the combined data, where DeltaG(u)(o) and DeltaG(w)(o) are the standard free energy changes of the N right harpoon over left harpoon D reaction in urea and water, respectively. Combination with the measured preferential binding gave the result that the increment of preferential binding, deltaGamma(23)=Gamma(23)(D)-Gamma(23)(N), is also linear in M(u). A temperature dependence study of preferential interactions permitted the evaluation of the transfer enthalpy, DeltaHmacr;(2,tr)(o), and entropy, DeltaSmacr;(2,tr)(o) of lysozyme from water into urea in both the native and denatured states. These values were found to be consistent with the enthalpy and entropy of formation of inter urea hydrogen bonds (Schellman, 1955; Kauzmann, 1959), with estimated values of DeltaHmacr;(2,tr)(o)=ca. -2.5 kcal mol(-1) and DeltaSmacr;(2,tr)(o)=ca. -7.0 e.u. per site. Analysis of the results led to the conclusion that the stabilization of the denatured form was predominantly by preferential binding to newly exposed peptide groups. Combination with the knowledge that stabilizing osmolytes act by preferential exclusion from peptide groups (Liu and Bolen, 1995) has led to the general conclusion that both the stabilization and destabilization of proteins by co-solvents are controlled predominantly by preferential interactions with peptide groups newly exposed on denaturation.     

Mechanism of the stabilization of ribonuclease A by sorbitol: preferential hydration is greater for the denatured then for the native protein.

The effect of interactions of sorbitol with ribonuclease A (RNase A) and the resulting stabilization of structure was examined in parallel thermal unfolding and preferential binding studies with the application of multicomponent thermodynamic theory. The protein was stabilized by sorbitol both at pH 2.0 and pH 5.5 as the transition temperature, Tm, was increased. The enthalpy of the thermal denaturation had a small dependence on sorbitol concentration, which was reflected in the values of the standard free energy change of denaturation, delta delta G(o) = delta G(o) (sorbitol) - delta G(o)(water). Measurements of preferential interactions at 48 degrees C at pH 5.5, where protein is native, and pH 2.0 where it is denatured, showed that sorbitol is preferentially excluded from the denatured protein up to 40%, but becomes preferentially bound to native protein above 20% sorbitol. The chemical potential change on transferring the denatured RNase A from water to sorbitol solution is larger than that for the native protein, delta mu(2D) > delta mu(2N), which is consistent with the effect of sorbitol on the free energy change of denaturation. The conformity of these results to the thermodynamic expression of the effect of a co-solvent on denaturation, delta G(o)(W) + delta mu(D)(2)delta G(o)(S) + delta mu(2D), indicates that the stabilization of the protein by sorbitol can be fully accounted for by weak thermodynamic interactions at the protein surface that involve water reversible co-solvent exchange at thermodynamically non-neutral sites. The protein structure stabilizing action of sorbitol is driven by stronger exclusion from the unfolded protein than from the native structure.     

Temperature dependence of the preferential interactions of ribonuclease A in aqueous co-solvent systems: thermodynamic analysis.

The temperature dependence of preferential solvent interactions with ribonuclease A in aqueous solutions of 30% sorbitol, 0.6 M MgCl2, and 0.6 M MgSO4 at low pH (1.5 and 2.0) and high pH (5.5) has been investigated. This protein was stabilized by all three co-solvents, more so at low pH than high pH (expect 0.6 M MgCl2 at pH 5.5). The preferential hydration of protein in all three co-solvents was high at temperatures below 30 degrees C and decreased with a further increase in temperature (for 0.6 M MgCl2 at pH 5.5, this was not significant), indicating a greater thermodynamic instability at low temperature than at high temperature. The preferential hydration of denatured protein (low pH, high temperature) was always greater than that of native protein (high pH, high temperature). In 30% sorbitol, the interaction passed to preferential binding at 45% for native ribonuclease A and at 55 degrees C for the denatured protein. Availability of the temperature dependence of the variation with sorbitol concentration of the chemical potential of the protein, (delta mu(2)/delta m3)T,p,m2, permitted calculation of the corresponding enthalpy and entropy parameters. Combination with available data on sorbitol concentration dependence of this interaction parameter gave (approximate) values of the transfer enthalpy, delta H2,tr, and transfer entropy delta S2,tr. Transfer of ribonuclease A from water into 30% sorbitol is characterized by positive values of the transfer free energy, transfer enthalpy, transfer entropy, and transfer heat capacity. On denaturation, the transfer enthalpy becomes more positive. This increment, however, is small relative to both the enthalpy of unfolding in water and to the transfer enthalpy of the native protein from water a 30% sorbitol solution.     

Molecular Dynamics/Free Energy Perturbation Studies of the Thermostable V74I Mutant of Ribonuclease HI

Abstract

Recent site-directed mutagenesis and thermodynamic studies have shown that the V74I mutant of Escherichia coli ribonuclease HI (RNase HI) is more stable than the wild type protein [Ishikawa et al., Biochemistry 32, 6171 (1993)]. In order to clarify the stabilization mechanism of this mutant, we calculated the free energy change due to the mutation Val 74→Ile in both the native and denatured states by free energy perturbations based on molecular dynamics (MD) simulations. We carried out inclusive MD simulations for the protein in water; i.e., fully solvated, no artificial constraints applied, and all long-range Coulomb interactions included. We found that the free energy of the mutant increased slightly relative to the wild type, in the native state by 1.60 kcal/mol, and in the denatured state by 2.25 kcal/mol. The unfolding free energy increment of the mutant (0.66 ± 0.19 kcal/mol) was in good agreement with the experimental value (0.6 kcal/mol). The hysteresis error in the free energy calculations, i.e., forward and reverse perturbations, was only ±0.19 kcal/mol. These results show that the V74I mutant is stabilized relative to the wild type by the increased free energy of the denatured state and not by a decrease in the free energy of the native state as had been proposed earlier based on the mutant X-ray structure. It was found that the stabilization was caused by a loss of solvation energy in the mutant denatured state and not by improved packing interactions inside the native protein.     

Interaction of lysozyme with dyes. II. Binding of bromophenol blue

The binding of lysozyme with bromophenol blue (BPB) at various dye concentrations and pH was carried out at 25 degrees C by equilibrium dialysis, ultraviolet (UV) difference and circular dichroism (CD) spectral techniques. Binding isotherms at pH 5.0 show non-cooperative binding at low dye concentrations, which change over to cooperative binding at higher concentrations indicating biphasic nature. However, binding isotherms at pH 7.0 and 9.0 show cooperative binding only, at all concentrations of the dye. The number of available binding sites decreases with the increase of pH. Gibbs free energy change, calculated on the basis of Wyman's binding potential concept, decreases with the increase of pH. Binding isotherms at pH 5.0 obtained at a lower temperature of 8 degrees C, also indicate the biphasic nature similar to those observed at 25 degrees C, but with a slight decreased strength of binding. The UV difference spectra of the complex do not show any distinct peaks in the 285 to 297 nm region eliminating any possible interaction of BPB with tryptophan and tyrosine residues of the lysozyme molecule. The CD spectra of lysozyme-BPB complex show a decrease in ellipticities with reference to native lysozyme in the near UV and far UV regions. This indicates that the lysozyme-BPB complex has a lower helical content probably due to the conformational changes induced into the native enzyme. The appearance of new positive peaks at 315 nm in the near UV region and at 592 nm in the visible region of the CD spectra may be due to the induced asymmetry into the BPB molecule as a result of its binding to a cationic residue (probably a lysine residue) of lysozyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Low-temperature unfolding of a mutant of phage T4 lysozyme. 2. Kinetic investigations

A disulfide-bridged variant of bacteriophage T4 lysozyme has been found to undergo a low- as well as high-temperature unfolding transition in guanidinium chloride [see Chen and Schellman (1989)]. The kinetics for this process have been followed for several temperatures, a range of guanidinium chloride concentrations, and a number of values of pH. Microscopic rate constants for protein unfolding and refolding were extracted from these data to explore the nature of the cold unfolding transition. The data were interpreted using transition-state theory. It was found that the Arrhenius energy is temperature dependent. The transition state is characterized by (1) a high energy and low entropy compared to the native state, (2) a heat capacity which is closer to the native state than to the unfolded state, and (3) a low exposure to solvent compared to the unfolded state, as judged by its interaction with guanidinium chloride. With increasing concentration of guanidinium chloride, the low-temperature unfolding rate increases strongly, and the refolding rate decreases very strongly.     

The thermodynamic analysis of protein stabilization by sucrose and glycerol against pressure-induced unfolding.

We have studied the reaction native left arrow over right arrow denatured for the 33-kDa protein isolated from photosystem II. Sucrose and glycerol have profound effects on pressure-induced unfolding. The additives shift the equilibrium to the left; they also cause a significant decrease in the standard volume change (DeltaV). The change in DeltaV was related to the sucrose and glycerol concentrations. The decrease in DeltaV varied with the additive: sucrose caused the largest effect, glycerol the smallest. The theoretical shift of the half-unfolding pressure (P1/2) calculated from the net increase in free energy by addition of sucrose and glycerol was lower than that obtained from experimental mea- surements. This indicates that the free energy change caused by preferential hydration of the protein is not the unique factor involved in the protein stabilization. The reduction in DeltaV showed a large contribution to the theoretical P1/2 shift, suggesting that the DeltaV change, caused by the sucrose or glycerol was associated with the protein stabilization. The origin of the DeltaV change is discussed. The rate of pressure-induced unfolding in the presence of sucrose or glycerol was slower than the refolding rate although both were significantly slower than that observed without any stabilizers.     

Thermodynamic characterization of cytochrome c at low pH. Observation of the molten globule state and of the cold denaturation process.

Several reports have pointed out the existence of intermediate states (both kinetic and equilibrium intermediate) between the native and the denatured states. The molten globule state, a compact intermediate state in which the secondary structure is formed but the tertiary structure fluctuates considerably, is currently being studied intensively because of its possible implication in the folding process of several proteins. We have examined the thermal stability of horse cytochrome c at low pH between 2.0 and 3.2 and different potassium chloride concentrations by absorbance of the Soret band, far and near-ultraviolet circular dichroism (u.v. c.d.) and tryptophan fluorescence using a multidimensional spectrophotometer. The concentration of potassium chloride ranged from 0 M to 0.5 M. The experimental thermal denaturation curves show that: (1) the helical content of cytochrome c remains stable at higher temperature when the concentration of salt is increased; whereas (2) the extent of ordering of the tertiary structure is weakly dependent on salt concentration; and (3) for cytochrome c, the stabilization of the molten globule state is induced by the binding of anions. Other salts such as NaCl, LiCl, potassium ferricyanide (K3Fe(CN)6) and Na2SO4 may also be used to stabilize the molten globule state. The thermodynamic analysis of the denaturation curves of c.d. at 222 nm and c.d. at 282 nm shows that, whereas a two-state (native and denatured) transition is observed at low-salt concentration, the far and near-u.v. c.d. melting curves of cytochrome c do not coincide with each other at high-salt concentration, and a minimum of three different thermodynamic states (IIb, intermediate or IIc, and denatured) is necessary to achieve a sufficient analysis. The intermediate state (called IIc) is attributed to the molten globule state because of its high secondary structure content and the absence of tertiary structure. Therefore, at low pH, cytochrome c is present in at least four states (native, IIb, IIc and denatured) depending on the salt concentration and temperature. The thermodynamic parameters, i.e. the Gibbs free energy differences (delta G), the enthalpy differences (delta H), the midpoint temperatures (Tm) of the transition (IIb in equilibrium intermediate (IIc in equilibrium denatured) are determined. We also give estimates of the heat capacity differences (delta Cp) from the temperature dependence of the enthalpy differences. The enthalpy change and the heat capacity difference of the IIc in equilibrium denatured transition are non-zero. The number of charges (protons or chloride anions) released upon transitions are determined by analysing the pH and chloride anion concentration dependence of the Gibbs free energy.(ABSTRACT TRUNCATED AT 400 WORDS)     

Thermodynamic stability of proteins in salt solutions: A comparison of the effectiveness of protein stabilizers

The denaturation of proteins by guanidine hydrochloride was studied in the presence of different concentrations of stabilizing salts, namely potassium phosphate, ammonium sulfate, and potassium acetate. The denaturation transition was followed by observing changes in the peptide circular dichroism atpH 7.0 and 25°C. From these results the free energy of stabilization for the process native denatured was determined. It was found that the stabilizing power of the anions increased in the order acetate < sulfate < phosphate, in agreement with the anionic lyotropic series. Ribonuclease A, which is known to have a site that can bind either a phosphate or a sulfate ion, showed a larger stabilization by these anions than that for lysozyme, pepsinogen, and myoglobin.     

In a staphylococcal nuclease mutant the side-chain of a lysine replacing valine 66 is fully buried in the hydrophobic core

The crystal structure of the staphylococcal nuclease mutant V66K, in which valine 66 is replaced by lysine, has been solved at 1.97 A resolution. Unlike lysine residues in previously reported protein structures, this residue appears to bury its side-chain in the hydrophobic core without salt bridging, hydrogen bonding or other forms of electrostatic stabilization. Solution studies of the free energy of denaturation, delta GH2O, show marked pH dependence and clearly indicate that the lysine residue must be deprotonated in the folded state. V66K is highly unstable at neutral pH but only modestly less stable than the wild-type protein at high pH. The pH dependence of stability for V66K, in combination with similar measurements for the wild-type protein, allowed determination of the pKa values of the lysine in both the denatured and native forms. The epsilon-amine of this residue has a pKa value in the denatured state of 10.2, but in the native state it must be 6.4 or lower. The epsilon-amine is thus deprotonated in the folded molecule. These values enabled an estimation of the epsilon-amine's relative change in free energy of solvation between solvent and the protein interior at 5.1 kcal/mol or greater. This implies that the value of the dielectric constant of the protein interior must be less than 12.8. Lysine is usually found with the methylene groups of its side-chain partly buried but is nevertheless considered a hydrophilic surface residue. It would appear that the high pKa value of lysine, which gives it a positive charge at physiological pH, is the primary reason for its almost exclusive confinement to the surface proteins. When deprotonated, this amino acid type can be fully incorporated into the hydrophobic core.     

Temperature dependence of histidine ionization constants in myoglobin

The standard enthalpy of ionization of six titratable histidines in horse metaquomyoglobin was determined by repeating proton NMR titrations as a function of temperature and using the van't Hoff relationship. It was found that deltaH degrees varies between 16 and 37 kJ mol(-1) in the protein, compared with a value of 29 kJ mol(-1) in free histidine. The standard entropy change was evaluated by combining the enthalpy and free energy changes derived from the pKa values. Although the entropy change could not be precisely and accurately obtained by this method, it could be established that it spans a wide range, from -60 to 0 J K(-1) mol(-1), about the value of -23 J K(-1) mol(-1) for the free histidine. The entropy change was used within the framework of enthalpy-entropy compensation to partition the solvation component from the standard thermodynamic quantities for each of the titrating residues. It was shown that the partitioning of the values in the protein is not readily understood in terms of solvent accessibility or electrostatic interactions. The contribution of solvation effects to the temperature response appeared to be significant only in the case of His-119 and His-48. The standard quantities were also used to explore the energetics of proton binding in the native state at temperatures below the onset of thermal denaturation.     

Perturbations of the denatured state ensemble: modeling their effects on protein stability and folding kinetics.

By considering the denatured state of a protein as an ensemble of conformations with varying numbers of sequence-specific interactions, the effects on stability, folding kinetics, and aggregation of perturbing these interactions can be predicted from changes in the molecular partition function. From general considerations, the following conclusions are drawn: (1) A perturbation that enhances a native interaction in denatured state conformations always increases the stability of the native state. (2) A perturbation that promotes a non-native interaction in the denatured state always decreases the stability of the native state. (3) A change in the denatured state ensemble can alter the kinetics of aggregation and folding. (4) The loss (or increase) in stability accompanying two mutations, each of which lowers (or raises) the free energy of the denatured state, will be less than the sum of the effects of the single mutations, except in cases where both mutations affect the same set of partially folded conformations. By modeling the denatured state as the ensemble of all non-native conformations of hydrophobic-polar (HP) chains configured on a square lattice, it can be shown that the stabilization obtained from enhancement of native interactions derives in large measure from the avoidance of non-native interactions in the D state. In addition, the kinetic effects of fixing single native contacts in the denatured state or imposing linear gradients in the HH contact probabilities are found, for some sequences, to significantly enhance the efficiency of folding by a simple hydrophobic zippering algorithm. Again, the dominant mechanism appears to be avoidance of non-native interactions. These results suggest stabilization of native interactions and imposition of gradients in the stability of local structure are two plausible mechanisms involving the denatured state that could play a role in the evolution of protein folding and stability.     

Stability analysis for the cavity-filling mutations of the Myb DNA-binding domain utilizing free-energy calculations

We have analyzed the effect of cavity-filling mutations on protein stability by means of free-energy calculations based on molecular dynamics simulations to identify the factors contributing to stability changes caused by the mutations. We have studied the DNA-binding domain of Myb, which has a cavity in one of three homologous repeat units, and analyzed a series of mutations with nonnatural and natural amino acids at a single site, which change the size of the cavity. We found that the calculated free-energy changes caused by the mutations are in excellent agreement with experimental data (correlation coefficient 0.98). The free-energy changes in the native and denatured states were independently compared with the unfolding free-energy change (deltadeltaG) and cavity-volume changes (deltaV), and it was found that deltadeltaG and deltaV correlate with the native-state free-energy changes but not with the denatured-state free-energy changes. Further analyses in terms of enthalpy and entropy show that compensation between entropy and enthalpy occurs in the denatured state but not in the native state. The main contribution to the native-state free energy was found to be van der Waals interactions associated with the cavity. We estimate that the decrease in free energy per methylene group, which results from filling the cavity, is about 2 to 3 kcal/mol. These results suggest that the stabilization of a protein by cavity-filling mutations be determined primarily by the free energy associated with the cavity volume in the native state.     

Comparison of temperature effect on the guanidine hydrochloride and urea denaturations of lysozyme

Guanidine hydrochloride (GdnHCl) and urea denaturations of lysozyme have been observed at various temperatures by measuring changes in fluorescence. Both transitions appear to be two state, with GdnHCl almost twice as effecitve a denaturant as urea for this protein. By plotting the denaturant concentrations at midpoint of the transition vs. the experimental temperature, it can be demonstrated that urea-denatured lysozyme does not obtain the degree of unfolding found in lysozyme denatured by GdnHCl.     

Interaction of calf skin collagen with glycerol: linked function analysis

Glycerol stabilizes the triple-helical structure of solubilized calf skin collagen. The equilibrium melting temperature of the protein increased linearly from 38.0 degrees C in AS buffer (0.01 M NaOAc and 0.02 M NaCl, pH 4.0) to 43.0 degrees C in AS and 6 M glycerol buffer. To understand the thermodynamic basis of this effect on the equilibrium melting temperature and the glycerol inhibition of collagen self-association, the preferential interactions of native and denatured calf skin collagens in AS buffer containing 1.5, 3, and 4.5 M glycerol were measured with a precision densimeter. The results indicated that native collagen binds glycerol preferentially whereas denatured collagen neither binds nor repels glycerol. The preferential binding of glycerol by native collagen, when interpreted in terms of the three-component solution thermodynamics, suggests that the surface interaction of native collagen with glycerol is energetically more favorable than its interaction with water. By use of the Wyman linked function, the negative chemical potential change of collagen derived from its preferential binding of glycerol can account for both the glycerol stabilization of the triple-helical structure of collagen and the inhibition of in vitro self-association of monomers into fibrils.     

Spectrofluorimetric detection of two states during melting of ribonuclease T1

The melting temperature of ribonuclease T1 was studied by the fluorescent method. It was shown that in the melting region the tryptophanyl fluorescence spectrum of the protein containing a single tryptophanyl is the sum of two simple spectra typical for tryptophanyl located in the hydrophobic environment and for tryptophanyl completely accessible to aqueous solvent, correspondingly. This implies the evidence of two forms of the protein, i.e. native (folded) and denatured (unfolded), in the transition region. No intermediate states were found in measured quantities. Therefore, ribonuclease T1 melting process corresponds to the two states model. The free energy of native structure stabilization of the protein at room temperature is delta G approximately equal to 37 kJ/mol.     

Determination of partial unfolding enthalpy for lysozyme upon interaction with dodecyltrimethylammonium bromide using an extended solvation model

The interactions of dodecyltrimethylammonium bromides (DTABs) with hen egg lysozyme have been investigated at pH = 7.0 and 27 degrees C in phosphate buffer by isothermal titration calorimetry. DTAB interacts endothermically and activate lysozyme. The endothermicity of the lysozyme-DTAB interaction is in marked contrast to the exothermic interactions between sodium dodecyl sulphate (SDS) and lysozyme which have been attributed to specific binding between the anionic sulphate head groups and cationic amino acid residues. The enthalpies of interaction between the cationic surfactant (DTAB) and lysozyme are dominated by the endothermic unfolding of the native structure followed by an exothermic solvation of the lysozyme-DTAB complex by the addition of extra DTAB. A new direct calorimetric method to follow protein denaturation, and the effect of surfactants on the stability of proteins was introduced. The extended solvation model was used to reproduce the enthalpies of lysozyme-DTAB interaction over the whole range of DTAB concentrations. The solvation parameters recovered from the new equation, attributed to the structural change of lysozyme and its biological activity. At low concentrations of DTAB, the binding is mainly electrostatic, with some simultaneous interaction of the hydrophobic tail with nearby hydrophobic patches on the lysozyme. These initial interactions presumably cause some protein unfolding and expose additional hydrophobic sites. The DTAB-induced denaturation enthalpy of lysozyme is 86.46 +/- 0.02 kJ mol(-1).     

Thermodynamic and kinetic examination of protein stabilization by glycerol

The effect of concentrated glycerol on the thermal transitions of chymotrypsinogen and ribonuclease has been examined by differential spectrophotometry at 293 and 287 mm, respectively. It was found that for both proteins addition of glycerol raises the transition temperature, the increase in Tm being greater for ribonuclease than for chymotrypsinogen. This increase in the free energy of denaturation appears to reflect primarily a decrease in the entropy change. Analysis in terms of the Wyman linkage equation shows that, for both proteins, the exclusion of glycerol from the protein domain increases on denaturation i.e., the chemical potential of glycerol becomes even more positive when the protein unfolds relative to the native structure. This provides the thermodynamic stabilization free energy. Results of the kinetic examination of the slow unfolding reaction are consistent with the concept that the preferential exclusion of glycerol is related, at least in part, to enhanced solvent ordering.     

Pressure-induced unfolding of lysozyme in aqueous guanidinium chloride solution.

The pressure-induced unfolding of lysozyme was investigated in an aqueous guanidinium chloride solution by means of ultraviolet spectroscopy. Assuming a two-state transition model, volume changes were calculated from the slope of free energy vs. pressure plots over a temperature range of 10 to 60 degrees C. Between 25 and 60 degrees C, almost constant volume changes were observed in the transition region, which was reflected in almost identical slopes of the free energy change vs. pressure plots. On the other hand, the different slopes were observed in the pressure dependence of free energy change at temperatures lower than 25 degrees C. These data were interpreted as suggesting that a two-state model is not appropriate at low temperature, but instead one or more intermediates are present under these conditions. The volume changes for unfolding became less negative at temperatures higher than 25 degrees C.     

Thermal features of the bovine serum albumin unfolding by polyethylene glycols.

Albumin showed very poor affinity for polyethylene glycol molecular weight (Mw) 1000 (30 M(-1)) and Mw 8000 (400 M(-1)) (PEG 1000 and PEG 8000). Polyethylene glycol of low Mw favours the ionization of the tyrosine (TYR) residues of albumin. Such variation might be a consequence of the change in dielectric constant at the domain of the protein by PEG binding. PEGs of high Mws stabilize the native compact state of human albumin showing negative preferential interaction with the protein. Interaction between PEGs and albumin is thermodynamically unfavourable, and becomes even more unfavourable for denatured proteins whose surface areas are larger than those of native ones leading to a stabilization of the unfolded state, which is manifested as a lowering of the thermal transition temperature. PEG 8000 perturbs the structure of the protein surface, partially modifying the layer of water and the microenvironment of the superficial aromatic residues (tryptophan, TRP and TYR) which is in agreement with the modifications of the UV spectrum of albumin by PEG 8000 and circular dichroism (CD) spectrum at high temperatures.