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In order to elucidate the contribution of charged residues to protein stabilization at temperatures of over 100 °C, we constructed many mutants of the CutA1 protein ( EcCutA1) from Escherichia coli. The goal was to see if one can achieve the same stability as for a CutA1 from hyperthermophile Pyrococcus horikoshii that has the denaturation temperature near 150 °C. The hydrophobic mutant of EcCutA1 ( Ec0VV) with denaturation temperature ( T d ) of 113.2 °C was used as a template for mutations. The highest T d of Ec0VV mutants substituted by a single charged residue was 118.4 °C. Multiple ion mutants were also constructed by combination of single mutants and found to have an increased thermostability. The highest stability of multiple mutants was a mutant substituted by nine charged residues that had a T d of 142.2 °C. To evaluate the energy of ion-ion interactions of mutant proteins, we used the structural ensemble obtained by a molecular dynamics simulation at 300 K. The T d of ionic mutants linearly increases with the increments of the computed energy of ion-ion interactions for ionic mutant proteins even up to the temperatures near 140 °C, suggesting that ion-ion interactions cumulatively contribute to the stabilization of a protein at high temperatures.

The refolding of cysteine-free pyrrolidone carboxyl peptidase (PCP-0SH) from a hyperthermophile is unusually slow. PCP-0SH is trapped in the denatured (D1) state at 4 °C and pH 2.3, which is different from the highly denatured state in the presence of concentrated denaturant. In order to elucidate the mechanism of the unusually slow folding, we investigated the structure of the D1 state using NMR techniques with amino acid selectively labeled PCP-0SH. The HSQC spectrum of the D1 state showed that most of the resonances arising from the 114-208 residues are broadened, indicating that conformations of the 114-208 residues are in intermediate exchange on the microsecond to millisecond time scale. Paramagnetic relaxation enhancement data indicated the lack of long-range interactions between the 1-113 and the 114-208 segments in the D1 state. Furthermore, proline scanning mutagenesis showed that the 114-208 segment in the D1 state forms a loosely packed hydrophobic core composed of α4- and α6-helices. From these findings, we conclude that the 114-208 segment of PCP-0SH folds into a stable compact structure with non-native helix-helix association in the D1 state. Therefore, in the folding process from the D1 state to the native state, the α4- and α6-helices become separated and the central β-sheet is folded between these helices. That is, the non-native interaction between the α4- and α6-helices may be responsible for the unusually slow folding of PCP-0SH.

In order to elucidate the stabilization mechanism of CutA1 from Pyrococcus horikoshii (PhCutA1) with a denaturation temperature of nearly 150 degrees C, GuHCl denaturation and heat denaturation were examined at neutral and acidic pHs. As a comparison, CutA1 proteins from Thermus thermophilus (TtCutA1) and Oryza sativa (OsCutA1) were also examined, which have lower optimum growth temperatures of 75 and 28 degrees C, respectively, than that (98 degrees C) of P. horikoshii. GuHCl-induced unfolding and refolding curves of the three proteins showed hysteresis effects due to an unusually slow unfolding rate. The midpoints of refolding for PhCutA1, TtCutA1 and OsCutA1 were 5.7 M, 3.3 M, and 2.3 M GuHCl, respectively, at pH 8.0 and 37 degrees C. DSC experiments with TtCutA1 and OsCutA1 showed that the denaturation temperatures were remarkably high, 112.8 and 97.3 degrees C, respectively, at pH 7.0 and that the good heat reversibility was amenable to thermodynamic analyses. At acidic pH, TtCutA1 showed higher stability to both heat and denaturant than PhCutA1. Combined with the data for DSC and denaturant denaturation, the unfolding Gibbs energy of PhCutA1 could be depicted as a function of temperature. It was experimentally revealed that (1) the unusually high stability of PhCutA1 basically originates from a common trimer structure of the three proteins, (2) the stability of PhCutA1 is superior to those of the other two CutA1s over all temperatures above 0 degrees C at neutral pH, due to the decrease in both enthalpy and entropy, and (3) ion pairs of PhCutA1 contribute to the unusually high stability at neutral pH.

The cysteine-free pyrrolidone carboxyl peptidase (PCP-0SH) from a hyperthermophile, Pyrococcus furiosus, can be trapped in the denatured state under nondenaturing conditions, corresponding to the denatured structure that exists in equilibrium with the native state under physiological conditions. The denatured state is the initial state (D1 state) in the refolding process but differs from the completely denatured state (D2 state) in the concentrated denaturant. Also, it has been found that the D1 state corresponds to the heat-denatured state. To elucidate the structural basis of the D1 state, H/D exchange experiments with PCP-0SH were performed at pD 3.4 and 4 degrees C. The results indicated that amide protons in the C-terminal alpha6-helix region hardly exchanged in the D1 state with deuterium even after 7 days, suggesting that the alpha6-helix (from Ser188 to Glu205) of PCP-0SH was stably formed in the D1 state. In order to examine the role of the alpha6-helix in folding and stability, H/D exchange experiments with a mutant, A199P, at position 199 in the alpha6-helix region were performed. The alpha6-helix region of A199P in the D1 state was partially unprotected, while some hydrophobic residues were protected against the H/D exchange, although these hydrophobic residues were unprotected in the wild-type protein. These results suggest that the structure of A199P in the D1 state formed a temporary stable denatured structure with a non-native hydrophobic cluster and the unstructured alpha6-helix. Both the stability and the refolding rate decreased by the substitution of Pro for Ala199. We can conclude that the native-like helix (alpha6-helix) of PCP-0SH is already constructed in the D1 state and is necessary for efficient refolding into the native structure and stabilization of PCP-0SH.

Pyrrolidone carboxyl peptidases (PCPs) from hyperthermophiles have a structurally conserved and completely buried Glu192 in the hydrophobic core; in contrast, the corresponding residue in the mesophile protein is a hydrophobic residue, Ile. Does the buried ionizable residue contribute to stabilization or destabilization of hyperthermophile PCPs? To elucidate the role of the buried glutamic acid in stabilizing PCP from hyperthermophiles, we constructed five Glu192 mutants of PCP-0SH (C142S/C188S, Cys-free double mutant of PCP) from Pyrococcus furiosus and examined their thermal and pH-induced unfolding and crystal structures and compared them with those of PCP-0SH. The stabilities of apolar (E192A/I/V) and polar (E192D/Q) mutants were less than PCP-0SH at acidic pH values. In the alkaline region, the mutant proteins, except for E192D, were more stable than PCP-0SH. The thermal stability data and theoretical calculations indicated an apparent pKa value > or = 7.3 for Glu192. Present results confirmed that the protonated Glu192 in PCP-0SH forms strong hydrogen bonds with the carbonyl oxygen and peptide nitrogen of Pro168. New intermolecular hydrogen bonds in the E --> A/D mutants were formed by a water molecule introduced into the cavity created around position 192, whereas the hydrogen bonds disappeared in the E --> I/V mutants. Structure-based empirical stability of mutant proteins was in good agreement with the experimental results. The results indicated that (1) completely buried Glu192 contributes to the stabilization of PCP-0SH because of the formation of strong intramolecular hydrogen bonds and (2) the hydrogen bonds by the nonionized and buried Glu can contribute more than the burial of hydrophobic groups to the conformational stability of proteins.

The three-dimensional structure of the bifunctional tryptophan synthase alpha(2)beta(2) complex from Pyrococcus furiosus was determined by crystallographic analysis. This crystal structure, with the structures of an alpha subunit monomer and a beta(2) subunit dimer that have already been reported, is the first structural set in which changes in structure that occur upon the association of the individual tryptophan synthase subunits were observed. To elucidate the structural basis of the stimulation of the enzymatic activity of each of the alpha and beta(2) subunits upon alpha(2)beta(2) complex formation, the conformational changes due to complex formation were analyzed in detail compared with the structures of the alpha monomer and beta(2) subunit dimer. The major conformational changes due to complex formation occurred in the region correlated with the catalytic function of the enzyme as follows. (1) Structural changes in the beta subunit were greater than those in the alpha subunit. (2) Large movements of A46 and L165 in the alpha subunit due to complex formation caused a more open conformation favoring the entry of the substrate at the alpha active site. (3) The major changes in the beta subunit were the broadening of a long tunnel through which the alpha subunit product (indole) is transferred to the beta active site and the opening of an entrance at the beta active site. (4) The changes in the conformations of both the alpha and beta subunits due to complex formation contributed to the stabilization of the subunit association, which is critical for the stimulation of the enzymatic activities.

When the tryptophan synthase alpha- and beta(2)-subunits combine to form the alpha(2)beta(2)-complex, the enzymatic activity of each subunit is stimulated by 1-2 orders of magnitude. To elucidate the structural basis of this mutual activation, it is necessary to determine the structures of the alpha- and beta-subunits alone and together with the alpha(2)beta(2)-complex. The crystal structures of the tryptophan synthase alpha(2)beta(2)-complex from Salmonella typhimurium (Stalpha(2)beta(2)-complex) have already been reported. However, the structures of the subunit alone from mesophiles have not yet been determined. The structure of the tryptophan synthase alpha-subunit alone from Escherichia coli (Ecalpha-subunit) was determined by an X-ray crystallographic analysis at 2.3 A, which is the first report on the subunits alone from the mesophiles. The biggest difference between the structures of the Ecalpha-subunit alone and the alpha-subunit in the Stalpha(2)beta(2)-complex (Stalpha-subunit) was as follows. Helix 2' in the Stalpha-subunit, including an active site residue (Asp60), was changed to a flexible loop in the Ecalpha-subunit alone. The conversion of the helix to a loop resulted in the collapse of the correct active site conformation. This region is also an important part for the mutual activation in the Stalpha(2)beta(2)-complex and interaction with the beta-subunit. These results suggest that the formation of helix 2'that is essential for the stimulation of the enzymatic activity of the alpha-subunit is constructed by the induced-fit mode involved in conformational changes upon interaction between the alpha- and beta-subunits. This also confirms the prediction of the conformational changes based on the thermodynamic analysis for the association between the alpha- and beta-subunits.

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.

It has been generally believed that polar residues are usually located on the surface of protein structures. However, there are many polar groups in the interior of the structures in reality. To evaluate the contribution of such buried polar groups to the conformational stability of a protein, nonpolar to polar mutations (L8T, A9S, A32S, I56T, I59T, I59S, A92S, V93T, A96S, V99T, and V100T) in the interior of a human lysozyme were examined. The thermodynamic parameters for denaturation were determined using a differential scanning calorimeter, and the crystal structures were analyzed by X-ray crystallography. If a polar group had a heavy energy cost to be buried, a mutant protein would be remarkably destabilized. However, the stability (Delta G) of the Ala to Ser and Val to Thr mutant human lysozymes was comparable to that of the wild-type protein, suggesting a low-energy penalty of buried polar groups. The structural analysis showed that all polar side chains introduced in the mutant proteins were able to find their hydrogen bond partners, which are ubiquitous in protein structures. The empirical structure-based calculation of stability change (Delta Delta G) [Takano et al. (1999) Biochemistry 38, 12698--12708] revealed that the mutant proteins decreased the hydrophobic effect contributing to the stability (Delta G(HP)), but this destabilization was recovered by the hydrogen bonds newly introduced. The present study shows the favorable contribution of polar groups with hydrogen bonds in the interior of protein molecules to the conformational stability.

To evaluate the contribution of the amino acid residues on the surface of a protein to its stability, a series of hydrophobic mutant human lysozymes (Val to Gly, Ala, Leu, Ile, Met, and Phe) modified at three different positions on the surface, which are located in the alpha-helix (Val 110), the beta-sheet (Val 2), and the loop (Val 74), were constructed. Their thermodynamic parameters of denaturation and crystal structures were examined by calorimetry and by X-ray crystallography at 100 K, respectively. Differences in the denaturation Gibbs energy change between the wild-type and the hydrophobic mutant proteins ranged from 4.6 to -9.6 kJ/mol, 2.7 to -1.5 kJ/mol, and 3.6 to -0.2 kJ/mol at positions 2, 74, and 110, respectively. The identical substitution at different positions and different substitutions at the same position resulted in different degrees of stabilization. Changes in the stability of the mutant proteins could be evaluated by a unique equation considering the conformational changes due to the substitutions [Funahashi et al. (1999) Protein Eng. 12, 841-850]. For this calculation, secondary structural propensities were newly considered. However, some mutant proteins were not adapted to the equation. The hydration structures around the mutation sites of the exceptional mutant proteins were affected due to the substitutions. The stability changes in the exceptional mutant proteins could be explained by the formation or destruction of the hydration structures. These results suggest that the hydration structure mediated via hydrogen bonds covering the protein surface plays an important role in the conformational stability of the protein.

Salt bridges play important roles in the conformational stability of proteins. However, the effect of a surface salt bridge on the stability remains controversial even today; some reports have shown little contribution of a surface salt bridge to stability, whereas others have shown a favorable contribution. In this study, to elucidate the net contribution of a surface salt bridge to the conformational stability of a protein, systematic mutant human lysozymes, containing one Glu to Gln (E7Q) and five Asp to Asn mutations (D18N, D49N, D67N, D102N, and D120N) at residues where a salt bridge is formed near the surface in the wild-type structure, were examined. The thermodynamic parameters for denaturation between pH 2.0 and 4.8 were determined by use of a differential scanning calorimeter, and the crystal structures were analyzed by X-ray crystallography. The denaturation Gibbs energy (DeltaG) of all mutant proteins was lower than that of the wild-type protein at pH 4, whereas there was little difference between them near pH 2. This is caused by the fact that the Glu and Asp residues are ionized at pH 4 but protonated at pH 2, indicating a favorable contribution of salt bridges to the wild-type structure at pH 4. Each contribution was not equivalent, but we found that the contributions correlate with the solvent inaccessibility of the salt bridges; the salt bridge contribution was small when 100% accessible, while it was about 9 kJ/mol if 100% inaccessible. This conclusion indicates how to reconcile a number of conflicting reports about role of surface salt bridges in protein stability. Furthermore, the effect of salts on surface salt bridges was also examined. In the presence of 0.2 M KCl, the stability at pH 4 decreased, and the differences in stability between the wild-type and mutant proteins were smaller than those in the absence of salts, indicating the compensation to the contribution of salt bridges with salts. Salt bridges with more than 50% accessibility did not contribute to the stability in the presence of 0.2 M KCl.

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.

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.

In globular proteins, there are intermolecular hydrogen bonds between protein and water molecules, and between water molecules, which are bound with the proteins, in addition to intramolecular hydrogen bonds. To estimate the contribution of these hydrogen bonds to the conformational stability of a protein, the thermodynamic parameters for denaturation and the crystal structures of five Thr to Val and five Thr to Ala mutant human lysozymes were determined. The denaturation Gibbs energy (DeltaG) of Thr to Val and Thr to Ala mutant proteins was changed from 4.0 to -5.6 kJ/mol and from 1.6 to -6.3 kJ/mol, respectively, compared with that of the wild-type protein. The contribution of hydrogen bonds to the stability (DeltaDeltaG(HB)) of the Thr and other mutant human lysozymes previously reported was extracted from the observed stability changes (DeltaDeltaG) with correction for changes in hydrophobicity and side chain conformational entropy between the wild-type and mutant structures. The estimation of the DeltaDeltaG(HB) values of all mutant proteins after removal of hydrogen bonds, including protein-water hydrogen bonds, indicates a favorable contribution of the intra- and intermolecular hydrogen bonds to the protein stability. The net contribution of an intramolecular hydrogen bond (DeltaG(HB[pp])), an intermolecular one between protein and ordered water molecules (DeltaG(HB[pw])), and an intermolecular one between ordered water molecules (DeltaG(HB[ww])) could be estimated to be 8. 5, 5.2, and 5.0 kJ/mol, respectively, for a 3 A long hydrogen bond. This result shows the different contributions to protein stability of intra- and intermolecular hydrogen bonds. The entropic cost due to the introduction of a water molecule (DeltaG(H)()2(O)) could be also estimated to be about 8 kJ/mol.

To further examine the contribution of hydrogen bonds to the conformational stability of the human lysozyme, six Ser to Ala mutants were constructed. The thermodynamic parameters for denaturation of these six Ser mutant proteins were investigated by differential scanning calorimetry (DSC), and the crystal structures were determined by X-ray analysis. The denaturation Gibbs energy (DeltaG) of the Ser mutant proteins was changed from 2.0 to -5.7 kJ/mol, compared to that of the wild-type protein. With an analysis in which some factors that affected the stability due to mutation were considered, the contribution of hydrogen bonds to the stability (Delta DeltaGHB) was extracted on the basis of the structures of the mutant proteins. The results showed that hydrogen bonds between protein atoms and between a protein atom and a water bound with the protein molecule favorably contribute to the protein stability. The net contribution of one intramolecular hydrogen bond to protein stability (DeltaGHB) was 8.9 +/- 2.6 kJ/mol on average. However, the contribution to the protein stability of hydrogen bonds between a protein atom and a bound water molecule was smaller than that for a bond between protein atoms.

To elucidate the energetic features of the anomalously high-level stabilization of a hyperthermophile pyrrolidone carboxyl peptidase (PfPCP) from a hyperthermophilic archaeon, Pyrococcus furiosus, equilibrium and kinetic studies of the guanidine hydrochloride (GuHCl)-induced unfolding and refolding were carried out with CD measurements at 220 nm in comparison with those from the mesophile homologue (BaPCP) from Bacillus amyloliquefaciens. The mutant protein of PfPCP substituted with Ser at both Cys142 and Cys188 (PfC142/188S) was used. The GuHCl unfolding for PfC142/188S and BaPCP was reversible. It was difficult to obtain the equilibrated unfolding curve of the hyperthermophile proteins at temperatures below 50 degreesC and pH 7, because of the remarkably slow rate of the unfolding. The unfolding for PfC142/188S attained equilibrium after 7 days at 60 degreesC, resulting in the coincidence between the unfolding and refolding curves. The Gibbs energy change of unfolding, DeltaGH2O (56.6 kJ/mol), for PfC142/188S at 60 degreesC and pH 7 was dramatically higher than that (7.6 kJ/mol) for BaPCP at 40 degreesC and pH 7. The unfolding and refolding kinetics for PfC142/188S and BaPCP at both 25 and 60 degreesC at pH 7 were approximated as a single exponential. The rate constant in water (kuH2O) of the unfolding reaction for PfC142/188S (1.6 x 10(-)15 s-1) at 25 degreesC and pH 7 was drastically reduced by 7 orders of magnitude compared to that (1.5 x 10(-)8 s-1) for BaPCP, whereas the refolding rates (krH2O) in water for PfC142/188S (9.3 x 10(-)2 s-1) and BaPCP (3.6 x 10(-)1 s-1) at 25 degreesC and pH 7 were similar. These results indicate that the greater stability of the hyperthermophile PCP was characterized by the drastically slow unfolding rate.

The contribution of hydrogen bonds to the conformational stability of human lysozyme was investigated by the combination of calorimetric and X-ray analyses of six Tyr --> Phe mutants. Unfolding Delta G and unfolding Delta H values of the Tyr --> Phe mutant proteins were changed by from +0.3 to -4.0 kJ/mol and from 0 to -16 kJ/mol, respectively, compared to those of the wild-type protein. The net contribution of a hydrogen bond at a specific site to stability (Delta Gwild/HB), considering factors affected by substitutions, was evaluated on the basis of X-ray structures of the mutant proteins. In the present study, one of six mutant proteins was suitable for evaluating the strength of the hydrogen bond. Delta Gwild/HB for the intramolecular hydrogen bond at Tyr124 was evaluated to be 7.5 kJ/mol. Results of the analysis of other mutants also suggest that hydrogen bonds of the hydroxyl group of Tyr, including the hydrogen bond with a water molecule, contribute to the stabilization of the human lysozyme.

The thermostability of methionine aminopeptidase from a hyperthermophile P. furiosus (PfMAP) was extremely high: the denaturation temperature was 106.2 degreesC at pH 10.2. To explore the contribution of electrostatic interaction to the superior thermostability of PfMAP, the thermostability of PfMAP was examined by differential scanning calorimetry (DSC) in various salt concentrations in the acidic region far from the isoelectric point of PfMAP. (1) In 20 mM glycine buffer, the DSC curve of PfMAP exhibited a single peak. Transition temperatures (Tm) were lowered with decreasing pH from 4 to 3. The heat denaturation of PfMAP was not reversible. (2) Denaturation enthalpy (DeltaH) measured at different pHs linearly correlated with Tm up to 102 degreesC, suggesting that the denaturation heat capacity (DeltaCp) for PfMAP is constant up to 100 degreesC. DeltaCp was estimated to be 0.82 J K-1 g-1. (3) In the presence of 10-100 mM KCl at pH 3.2, two peaks appeared on the DSC curves. The first peak shifted to lower temperatures with increasing concentration of KCl and, oppositely, the second one to higher temperatures. It was found that the first and second peaks originated from the heat denaturation of the native form of PfMAP and the melting of the non-native associated form having molten globule-like structure, respectively, judged from the CD spectra and ultracentrifugation analyses. This indicates the following: first, the attractive electrostatic interaction is an important factor in stabilizing the native form of PfMAP; second, the presence of KCl stimulates the formation of the molten globule-like state of PfMAP and stabilizes it. (4) In a comparison of the sequence and crystal structure of PfMAP, which has been recently determined (1xgs.pdb), with those of MAP from Escherichia coli (EcMAP), it was predicted that the extra four short-range ion pairs less than 3 A involved in PfMAP are crucial candidates as determinants for the superior thermostability of PfMAP.

To elucidate the role of conserved proline residues of the tryptophan synthase alpha subunit from Escherichia coli in stability and folding, equilibrium and kinetic studies of the unfolding-refolding induced by guanidine hydrochloride for six mutant alpha subunits (Pro-->Ala) were carried out by peptidyl circular dichroism and aromatic fluorescence measurements at pH 7 and 25 degrees C. These results were analyzed assuming the presence of one intermediate (I) state in the denaturation process. (I) For all mutant and wild-type proteins, the Gibbs energy change (delta Gni(H2O)) in water between the native (N) and I states coincided with the difference (delta G++u(H2O)-delta G++r(H2O)) between the activation Gibbs energy changes in water for the unfolding (delta G++u(H2O) and refolding (delta G++r(H2O) reactions. This means that the early folding intermediate of the alpha subunit corresponds to the equilibrium intermediate. Delta Gni(H2O) values of all mutant proteins decreased compared with that of the wild-type protein. Gibbs energy change (delta Gid(H2O) in water between I and the denatured (D) states was not substantially affected by the substitutions. Delta G++u(H2O) and delta G++r(H2O) decreased and increased, respectively, for all mutant proteins. (2) Six conserved prolines played roles in stability and folding of the alpha subunit in a different manner: prolines 28 and 96 by stabilizing the N state and prolines 28, 96, 132, and 207 by destabilizing the I state. The contributions of prolines 57 and 62 to the stability were marginal. (3) Cis proline 28 was not the origin of the slow phase in the refolding kinetics assumed to arise from the cis-trans isomerization reaction of proline.

To clarify the contribution of the hydrophobic effect to the conformational stability of human lysozyme, a series of Val to Ala mutants were constructed. The thermodynamic parameters for the denaturation of these nine mutant proteins were determined using differential scanning calorimetry (DSC), and the crystal structures were solved at high resolution. The denaturation Gibbs energy (delta delta G) and enthalpy (delta delta H) values of the mutant proteins ranged from +2.2 to- 6.3 kJ/mol and from +7 to -17 kJ/mol, respectively. The structural analyses showed that the mutation site and/or the residues around it in some proteins shifted toward the created cavity, and the substitutions affected not only the mutations site but also other parts far from the site, although the structural changes were not as great. Correlation between the changes in the thermodynamic parameters and the structural features of mutant proteins was examined, including the five Ile to Val mutant human lysozymes [Takano et al. (1995) J. Mol. Biol. 254, 62-76]. There was no simple general correlation between delta delta G and the changes in hydrophobic surface area exposed upon denaturation (delta delta ASAHP). We found only a new correlation between the delta delta G and delta delta ASAHP of all of the hydrophobic residues if the effect of the secondary structure propensity was taken into account.

The unfolding and refolding processes of Escherichia coli ribonuclease HI at 25 degrees C, induced by concentration jumps of either guanidine hydrochloride (GuHCl) or urea, were investigated using stopped-flow circular dichroism (CD), stopped-flow fluorescence, and NMR spectroscopies. Only a single exponential process was detected for the fast time scale unfolding (rate constants from 0.014 to 0.54 s-1, depending on the final denaturant concentration). For refolding, the far-UV CD value largely recovered within 50 ms of the stopped-flow mixing dead time (burst phase). This phase was followed by either one or two phases, with rate constants from 0.035 to 2.45 s-1 as detected by CD and fluorescence, respectively. Although this protein has a single cis-Pro residue, a very slow phase due to proline isomerization was not observed, for either unfolding or refolding. The difference in the amplitudes of the burst phases for refolding in the far- and near-UV CD spectra revealed that an intermediate state exists, with the characteristics of a molten globule. Because the one-phased fast exponential process detected by CD corresponds to the slower of the two phases detected by fluorescence, the intermediate detected by CD might be the most stable. GuHCl denaturation experiments revealed that this intermediate cooperatively unfolds, with a transition midpoint of 1.33 +/- 0.03 M. The Gibbs free energy difference (delta G) between the intermediate and the unfolded states, under physiological conditions (25 degrees C, pH 5.5, and 0 M GuHCl), was estimated to be 20.0 +/- 2.3 kJ mol-1. Therefore, it is reasonable to assume that the refolding intermediate, rather than the unfolded state, is the latent denatured state under physiological conditions. Approximately linear relationships between the GuHCl concentration and the logarithm of the microscopic rate constants determined by CD and fluorescence were also observed. By extrapolation to a GuHCl concentration of 0 M, activation Gibbs free energies of 98.5 +/- 1.1 kJ mol-1 for unfolding and 69.5 +/- 0.2 kJ mol-1 for refolding under physiological conditions were obtained. The hydrogen-exchange-refolding competition combined with two-dimensional NMR revealed that the amide protons of alpha-helix I are the most highly protected, suggesting that alpha-helix I is the initial site of protein folding. The CD and NMR data showed that the intermediate state has a structure similar to that of the acid-denatured molten globule.

To understand the role of disulfide bridges in protein stability, the thermodynamic changes in the denaturation of two mutant human lysozymes lacking a disulfide bridge between Cys-77 and Cys-95 (C77A and C77/95A) were analyzed using differential scanning calorimetry (DSC). At pH 3.0 and 57 degrees C, the stabilities of both the C77A and C77/95A mutants were decreased about 4.6 kcal.mol-1 in Gibbs free energy change. Under the same conditions, the enthalpy changes (delta H) were 94.8 and 90.8 kcal.mol-1, respectively, which were smaller than that of the wild type (100.8 kcal.mol-1). The destabilization of the mutants was caused by enthalpic factors. Although X-ray crystallography indicated that the mutants preserve the wild-type tertiary structure, removal of the disulfide bridge increased the flexibility of the native state of the mutants. This was indicated both by an increase in the crystallographic thermal factors (B-factors) and by a decrease in the affinity of N-acetylglucosamine trimer [(NAG)3] observed using isothermal titration calorimetry (DTC) due to entropic effects. Thus, the effect of cross-linking on the stability of a protein is not solely explained by the entropy change in denaturation.

It has been shown that protein stability can be modulated from site-directed mutations that affect the entropy of protein unfolding [Matthews, B. W., Nicholson, H., & Becktel, W. J. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 6663-6667]. However, the effect of a specific amino acid replacement on stability highly depends on the location of the mutation site and its environment in the protein structure [Yutani, K., Hayashi, S., Sugisaki, Y., & Ogasahara, K. (1991) Proteins Struct., Funct., Genet. 9, 90-98). To clarify the role of specific proline residues in the thermostability of human lysozyme (h-lysozyme), a series of proline mutants were investigated by means of scanning calorimetry and high-resolution X-ray crystallography. The thermodynamic properties of the mutant and wild-type h-lysozymes are compared and discussed on the basis of their three-dimensional structure. h-Lysozyme contains two proline residues at positions 71 and 103. The Pro71----Gly substitution was found to destabilize h-lysozyme by decreasing the entropic contribution of unfolding by about 2 kcal/mol at 68.8 degrees C. This is consistent with the theoretical expectations for such a substitution. However, the same substitution at position 103 (Pro103----Gly) does not affect h-lysozyme stability, and the thermodynamic properties of the P71G/P103G and P71G mutants are essentially the same. Pro71 which is conserved among lysozymes from other species, appears to be important for stability, whereas Pro103, which is not conserved, does not. These differences are explained in terms of residue accessibility to the solvent and crystallographic B-factor, which reflects the amino acid mobility.(ABSTRACT TRUNCATED AT 250 WORDS)

The unfolding and refolding kinetics of six proline mutants of the human lysozyme (h-lysozyme) were carried out and compared to that of the wild-type protein. Our results show that the slow refolding phase observed in the h-lysozyme refolding kinetics cannot be ascribed to proline isomerization reactions. The h-lysozyme contains two proline residues at positions 71 and 103, both in the trans conformation in the native state. The refolding kinetics of the P71G/P103G mutant, in which both prolines have been replaced by a glycine, were found to be similar to those of the wild-type protein. The same slow phase amplitude of about 10% was found for both proteins, and the slow phase rate constants were also identical within experimental error. Other mutants such as P103G or P71G, in which only one of the two prolines has been replaced by a glycine, and A47P with its three prolines, gave identical slow refolding phases. The X-ray structure analysis and scanning microcalorimetric study of each protein (Herning et al., unpublished experiments) have confirmed that none of the considered mutations affects significantly protein structure and that no major changes in protein stability were brought about by these mutations. Therefore, comparison of the properties of the mutant and wild-type proteins is legitimate. Interestingly, the refolding kinetics of the V110P mutant, in which a proline residue has been introduced at position 110 (N-terminus of an alpha-helix), were clearly triphasic. For this mutant an additional very slow phase with properties similar to those expected from the proline hypothesis was detected. Equilibrium denaturation studies were conducted for each protein, and the refolding pathway of h-lysozyme is partly presented. We also discuss the effect of proline mutations on the energetics of the folding pathway of the h-lysozyme in water.

The relationship between the domain structure of the alpha subunit of Escherichia coli tryptophan synthase and the mechanism of unfolding of the alpha subunit is investigated. Previous studies of the unfolding of the alpha subunit by increasing concentrations of guanidine hydrochloride or urea detected a partially unfolded form of the alpha subunit at intermediate concentrations of either denaturant. The possibility that this partially unfolded form of the alpha subunit results from the preferential unfolding of one of the two domains of the alpha subunit is now investigated. This study utilizes two proteolytic fragments of the alpha subunit, alpha-1 and alpha-2, which have been shown to refold independently and to correspond to two domains of the alpha subunit. The effects of guanidine hydrochloride concentration on the separate alpha-1 and alpha-2 fragments, on the intact alpha subunit, and on the derivative nicked by trypsin (alpha') are compared by measuring ellipticity at 222 nm and by measuring the susceptibility of tyrosyl residues to chemical modification. The results show that guanidine hydrochloride induced unfolding of the alpha subunit results from the stepwise unfolding of the two domains: the alpha-2 fragment and the corresponding domain in the intact alpha subunit are unfolded by low concentrations of guanidine hydrochloride; the alpha-1 fragment and the corresponding domain in the intact alpha subunit are unfolded by higher concentrations of guanidine hydrochloride.

In order to elucidate the effect of single amino acid substitutions on the conformation of the tryptophan synthase alpha-subunit from Escherichia coli in solution, 1H NMR spectra of the wild-type and mutant proteins were measured at various pHs. Two of the four His C2-proton resonances of the alpha-subunit were assigned to two His residues at positions 92 and 146 by using a mutant protein with Thr substituted for the His at position 92. The replacement did not affect the conformation of the protein significantly. The proton resonances of all the Tyr residues in the aromatic region could be picked up from other resonance peaks, employing the wild-type alpha-subunit deuterated at all of the Phe residues. On comparison of the spectra of the wild-type protein with those of the mutant protein with Met substituted for the Glu at position 49, it was concluded that the substitution affects only the residues close to the substituted residue at acidic pH but that a larger part of the protein is affected at alkaline pH. NOE experiments showed that the five Tyr residues, four of which are located in the proximity of position 49, are close to one another. The present results are discussed in the light of the conformational stability of the protein.