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K Gekko

Publications and source records attributed to K Gekko.

At least 19 recordsLinked to original sources
PubMed2001

Effects of five-tryptophan mutations on structure, stability and function of Escherichia coli dihydrofolate reductase.

To elucidate the roles of tryptophan residues in the structure, stability, and function of Escherichia coli dihydrofolate reductase (DHFR), its five tryptophan residues were replaced by site-directed mutagenesis with leucine, phenylalanine or valine (W22F, W22L, W30L, W47L, W74F, W74L, W133F, and W133V). Far-ultraviolet circular dichroism (CD) spectra of these mutants reveal that exciton coupling between Trp47 and Trp74 strongly affects the peptide CD of wild-type DHFR, and that Trp133 also contributes appreciably. No additivity was observed in the contributions of individual tryptophan residues to the fluorescence spectrum of wild-type DHFR, Trp74 having a dominant effect. These single-tryptophan mutations induce large changes in the free energy of urea unfolding, which showed values of 1.79-7.14 kcal/mol, compared with the value for wild-type DHFR of 6.08 kcal/mol. Analysis of CD and fluorescence spectra suggests that thermal unfolding involves an intermediate with the native-like secondary structure, the disrupted Trp47-Trp74 exciton coupling, and the solvent-exposed Trp30 and Trp47 side chains. All the mutants except W22L (13%) retain more than 50% of the enzyme activity of wild-type DHFR. These results demonstrate that the five tryptophan residues of DHFR play important roles in its structure and stability but do not crucially affect its enzymatic function.

Circular Dichroism↗
PubMed2000

High pressure NMR reveals active-site hinge motion of folate-bound Escherichia coli dihydrofolate reductase.

A high-pressure (15)N/(1)H two-dimensional NMR study has been carried out on folate-bound dihydrofolate reductase (DHFR) from Escherichia coli in the pressure range between 30 and 2000 bar. Several cross-peaks in the (15)N/(1)H HSQC spectrum are split into two with increasing pressure, showing the presence of a second conformer in equilibrium with the first. Thermodynamic analysis of the pressure and temperature dependencies indicates that the second conformer is characterized by a smaller partial molar volume (DeltaV = -25 mL/mol at 15 degrees C) and smaller enthalpy and entropy values, suggesting that the second conformer is more open and hydrated than the first. The splittings of the cross-peaks (by approximately 1 ppm on (15)N axis at 2000 bar) arise from the hinges of the M20 loop, the C-helix, and the F-helix, all of which constitute the major binding site for the cofactor NADPH, suggesting that major differences in conformation occur in the orientations of the NADPH binding units. The Gibbs free energy of the second, open conformer is 5.2 kJ/mol above that of the first at 1 bar, giving an equilibrium population of about 10%. The second, open conformer is considered to be crucial for NADPH binding, and the NMR line width indicates that the upper limit for the rate of opening is 20 s(-)(1) at 2000 bar. These experiments show that high pressure NMR is a generally useful tool for detecting and analyzing "open" structures of a protein that may be directly involved in function.

Binding Sites↗
PubMed2000

Effect of ligand binding on the flexibility of dihydrofolate reductase as revealed by compressibility.

The partial specific volume, v, and adiabatic compressibility, beta(s), of Escherichia coli dihydrofolate reductase were measured at 30 degrees C in the presence of various ligands (folate, dihydrofolate, tetrahydrofolate, NADPH, NADP, methotrexate, and KCl). Binding of these ligands (binary and ternary complexes) brought about large changes of v (0.734-0.754 cm(3) g(-1)) and beta(s) (6. 6x10(-6)-9.8x10(-6) bar(-1)), keeping a linear relationship between the two parameters. The values of v and beta(s) increased with an increase in internal cavity, V(cav), and a decrease in accessible surface area, ASA, which were calculated from the X-ray crystal structures of the complexes. A large variation of V(cav) relative to ASA by ligand binding suggested that the cavity is a dominant factor and the effect of hydration might be small for the ligand-induced changes of v and beta(s). The beta(s) values of the binary and ternary complexes suggested a characteristic conformational flexibility of the kinetic intermediates in the enzyme reaction coordinate. Comparison of beta(s) with the cavity distribution in the crystal structures revealed that the flexibility of the intermediates was mainly determined by the total cavity volume with minor contributions of the number, position, and size of cavities. These results demonstrate that the compressibility is a useful measure of the conformational flexibility of the intermediates in the enzyme reaction and that the combined study of compressibility and X-ray crystallography gives new insight into the protein dynamics through the behavior of the cavities.

Escherichia coli↗
PubMed2000

Nitrilase of Rhodococcus rhodochrous J1. Conversion into the active form by subunit association.

Nitrilase-containing resting cells of Rhodococcus rhodochrous J1 converted acrylonitrile and benzonitrile to the corresponding acids, but the purified nitrilase hydrolyzed only benzonitrile, and not acrylonitrile. The activity of the purified enzyme towards acrylonitrile was recovered by preincubation with 10 mM benzonitrile, but not by preincubation with aliphatic nitriles such as acrylonitrile. It was shown by light-scattering experiments, that preincubation with benzonitrile led to the assembly of the inactive, purified and homodimeric 80-kDa enzyme to its active 410-kDa aggregate, which was proposed to be a decamer. Furthermore, the association concomitant with the activation was reached after dialysis of the enzyme against various salts and organic solvents, with the highest recovery reached at 10% saturated ammonium sulfate and 50% (v/v) glycerol, and by preincubation at increased temperatures or enzyme concentrations.

Acrylonitrile↗
PubMed2000

Single amino acid substitutions in flexible loops can induce large compressibility changes in dihydrofolate reductase.

To address the effects of single amino acid substitutions on the flexibility of Escherichia coli dihydrofolate reductase (DHFR), the partial specific volume (v(o)) and adiabatic compressibility (beta(s)(o)) were determined for a series of mutants with amino acid replacements at Gly67 (7 mutants), Gly121 (6 mutants), and Ala145 (5 mutants) located in three flexible loops, by means of precise sound velocity and density measurements at 15 degrees C. These mutations induced large changes in v(o) (0.710-0.733 cm(3). g(-1)) and beta(s)(o) (-1.8 x 10(-6)-5.5 x 10(-6) bar(-1)) from the corresponding values for the wild-type enzyme (v(o)=0.723 cm(3). g(-1), beta(s)(o) = 1.7 x 10(-6) bar(-1)), probably due to modifications of internal cavities. The beta(s)(o) value increased with increasing v(o), but showed a decreasing tendency with the volume of the amino acid introduced. There was no significant correlation between beta(s)(o) and the overall stability of the mutants determined from urea denaturation experiments. However, a mutant with a large beta(s)(o) value showed high enzyme activity mainly due to an enhanced catalytic reaction rate (k(cat)) and in part due to increased affinity for the substrate (K(m)), despite the fact that the mutation sites are far from the catalytic site. These results demonstrate that the flexibility of the DHFR molecule is dramatically influenced by a single amino acid substitution in one of these loops and that the flexible loops of this protein play important roles in determining the enzyme function.

Amino Acid Substitution↗
PubMed1999

Polyol-induced molten globule of cytochrome c: an evidence for stabilization by hydrophobic interaction.

To address the contribution of hydrophobic interaction to the stability of molten globule (MG) of proteins, the effects of various polyols (ethylene glycol, glycerol, erythritol, xylitol, sorbitol, and inositol) on the structure of acid-unfolded horse cytochrome c were examined at pH 2, by means of circular dichroism (CD), partial specific volume, adiabatic compressibility, and differential scanning calorimetry (DSC). Addition of polyols induced the characteristic CD spectra of MG, the effect being enhanced with an increase in their concentration and chain length (the number of OH groups) of polyols except for ethylene glycol. The free energy change of MG formation by sorbitol was comparable with those for the salt-induced MG formation but the heat capacity change was negligibly small. The partial specific volume did not change within the experimental error but the adiabatic compressibility largely increased by MG formation. The sorbitol-induced MG showed a highly cooperative DSC thermogram with a large heat capacity change in comparison with the salt-induced one. These results demonstrate that polyols can stabilize the MG state of this protein through the enhanced hydrophobic interaction overcoming the electrostatic repulsion between charged residues. The stabilizing mechanism and structure of MG state induced by polyols were discussed in terms of the preferential solvent interactions and osmotic pressure of the medium, in comparison with the salt-induced one.

Calorimetry, Differential Scanning↗
PubMed1998

Acetonitrile-protein interactions: amino acid solubility and preferential solvation.

The solubility of amino acids and the preferential solvent interaction of hen-egg lysozyme in acetonitrile (AN)-water mixtures (<60 w/v% AN) were investigated by means of densimetric and refractometric methods at 25 degreesC. The free energy of transfer from water to aqueous AN was negative for most nonpolar side-chains of amino acids and positive for the peptide group, the extent being comparable to those for methanol and ethanol systems. Addition of AN to an aqueous solvent was thus suggested to weaken the hydrophobic interaction and to enhance the peptide-peptide hydrogen bond therein leading to the denaturation of proteins. A parallel examination by circular dichroism confirmed that the conformation of lysozyme (pH 3) remains native in aqueous AN up to 40% but changes to the helix-rich form at higher AN concentrations. At all solvent compositions up to 50% AN (pH 3), however, lysozyme was preferentially hydrated probably due to a local salting-out of the AN molecules from the charges on the protein surface, indicating the increase of the chemical potential of the protein. These results are discussed in relation to the role of AN as an eluting organic solvent in reverse-phase chromatography.

Acetonitriles↗
PubMed1998

Nonadditive effects of double mutations at the flexible loops, glycine-67 and glycine-121, of Escherichia coli dihydrofolate reductase on its stability and function.

The structure, stability, and enzymatic function of dihydrofolate reductase (DHFR) from Escherichia coli are influenced by point mutations at sites 67 and 121 in two flexible loops [Gekko et al. (1994) J. Biochem. 116, 34-41; Ohmae et al. (1996) J. Biochem. 119, 703-710]. In the present study, eight double mutants at sites 67 and 121 (G67V/G121S, G67V/G121A, G67V/G121C, G67V/G121D, G67V/G121V, G67V/G121H, G67V/G121L, and G67V/G121Y) were constructed in order to identify interactions between the two sites of DHFR. The far-ultraviolet circular dichroism spectra of double mutants were clearly different from those of the respective single mutants, with significant changes being observed for three mutants, G67V/G121A, G67V/G121L, and G67V/G121S. The Gibbs free energy change of urea unfolding of double mutants could not be expressed by the sum of those of the respective single mutants except for G67V/G121H. The steady-state kinetic experiments showed that the effect of double mutations manifests itself not in Km but in k(cat), and the transition-state stabilization energy for G67V/G121A, G67V/G121C, and G67V/G121L is not equal to the sum of those for the single mutants. These results indicate that the additivity rule essentially does not hold for these double mutants, and that long-range interactions occur between sites 67 and 121, even though they are separated by 27.7 A. This is evidence that the flexible loops play important roles in the stability and function of this enzyme through structural perturbations, in which a small alteration in local atomic packing due to amino acid substitution is cooperatively magnified over almost the whole molecule.

Circular Dichroism↗
PubMed1998

Effects of point mutations at the flexible loop alanine-145 of Escherichia coli dihydrofolate reductase on its stability and function.

To elucidate the role of a flexible loop (residues 142-149) in the stability and function of Escherichia coli dihydrofolate reductase, alanine-145 in this loop was substituted by site-directed mutagenesis with ten amino acids (Glu, Phe, Gly, His, Ile, Leu, Arg, Ser, Thr, and Val). The amount of three mutant proteins (A145E, A145I, and A145L) in cells was too small to allow the measurement of circular dichroism (CD) spectra and urea unfolding. The CD spectra of other seven mutants were identical with those of the wild-type DHFR, indicating that the native conformation of DHFR was not affected by the mutations. The free energy change of unfolding by urea decreased with an increase in the hydrophobicity of amino acid residues introduced, A145T>A145R>A145G>=A145S>=A145H>A145V++ +>wild-type>=A145F. The steady-state kinetic parameters for the enzyme reaction, Km and ksub, were only slightly influenced by the mutations. These results suggest that site 145 in the flexible loop plays an important role in the stability but has little or no effect on the native structure and function of this enzyme. The characteristics of the mutations are discussed in comparison with those of mutations at site 67 [Ohmae et al. (1996) J. Biochem. 119, 703-710] and at site 121 [Gekko et al. (1994) J. Biochem. 116, 34-41] in two other flexible loops.

Alanine↗
PubMed1998

Protective roles of bacterioruberin and intracellular KCl in the resistance of Halobacterium salinarium against DNA-damaging agents.

Halobacterium salinarium, a member of the extremely halophilic archaebacteria, contains a C50-carotenoid namely bacterioruberin. We have previously reported the high resistance of this organism against the lethal actions of DNA-damaging agents including ionizing radiation and ultraviolet light (UV). In this study, we have examined whether bacterioruberin and the highly concentrated salts in this bacterium play protective roles against the lethal actions of ionizing radiation, UV, hydrogen peroxide, and mitomycin-C (MMC). The colourless mutant of H. salinarium deficient in bacterioruberin was more sensitive than the red-pigmented wild-type to all tested DNA-damaging agents except MMC. Circular dichroism (CD) spectra of H. salinarium chromosomal DNA at various concentrations of KCl (0-3.5 M) were similar to that of B-DNA, indicating that no conformational changes occurred as a result of high salt concentrations. However, DNA strand-breaks induced by ionizing radiation were significantly reduced by the presence of either bacterioruberin or concentrated KCl, presumably due to scavenging of free radicals. These results suggest that bacterioruberin and intracellular KCl of H. salinarium protect this organism against the lethal effects of oxidative DNA-damaging agents.

Carotenoids↗
PubMed1997

Adiabatic compressibility of flagellin and flagellar filament of Salmonella typhimurium.

The partial specific volume and adiabatic compressibility of flagellin, its F40 fragment deprived of the disordered terminal regions, from Ala-1 to Arg-65 and from Ser-451 to Arg-494, and the flagellar filament of Salmonella typhimurium were determined from the density and the sound velocity measurements at 15 degrees C. The partial specific volumes were 0.728 cm3/g, 0.745 cm3/g, and 0.734 cm3/g, and the partial specific adiabatic compressibilities were 4.0 x 10(-12) cm2/dyn, 6.7 x 10(-12) cm2/dyn, and 4.7 x 10(-12) cm2/dyn, for flagellin, F40, and the filament, respectively. The smaller values of flagellin than those of F40 are reasonably explained by the presence of disordered terminal regions, which are supposed to be highly hydrated by water molecules. The volume increase upon polymerization of flagellin into the filament is also confirmed by depolymerization under a high pressure. The smaller volume and compressibility of the filament compared with those of F40 suggest an extensive hydration of the filament on its complex surface structure, which surpasses the effect on the volume and compressibility by a possible increase in the cavity volume at intersubunit interfaces upon polymerization.

Circular Dichroism↗
PubMed1997

High-salt effects on the structure and damage of chromosomal DNA in Halobacterium salinarium, an extremely halophilic bacterium.

High concentration salt effects on the structure and radiation-induced damages of DNA were studied to elucidate the biochemical mechanism of the resistance of halophilic H. salinarium against DNA damaging agents. High concentration of KCl did not induce significant conformational changes in H. salinarium chromosomal DNA, but exhibited an extensive protective effect on the radiation-induced single-strand breaks of plasmid DNA.

Chromosomes, Bacterial↗
PubMed1996

A large compressibility change of protein induced by a single amino acid substitution.

The adiabatic compressibility (beta s) was determined, by means of the precise sound velocity and density measurements, for a series of single amino acid substituted mutant enzymes of Escherichia coli dihydrofolate reductase (DHFR) and aspartate aminotransferase (AspAT). Interestingly, the beta s values of both DHFR and AspAT were influenced markedly by the mutations at glycine-121 and valine-39, respectively, in which the magnitude of the change was proportional to the enzyme activity. This result demonstrates that the local change of the primary structure plays an important role in atomic packing and protein dynamics, which leads to the modified stability and enzymatic function. This is the first report on the compressibility of mutant proteins.

Aspartate Aminotransferases↗
PubMed1996

Effects of point mutations at the flexible loop glycine-67 of Escherichia coli dihydrofolate reductase on its stability and function.

To elucidate the role of a flexible loop (residues 64-72) in the stability and function of Escherichia coli dihydrofolate reductase, glycine-67 in this loop was substituted by site-directed mutagenesis with seven amino acids (Ala, Cys, Asp, Leu, Ser, Thr, and Val). The circular dichroism spectra suggested that the confirmation of the native structure was affected by the mutations in both the presence and absence of NADPH. The free energy change of unfolding by urea decreased in the order of G67A > G67S > or = wild-type > or = G67D > G67T > G67C > or = G67L > G67V. The steady-state kinetic parameters for the enzyme reaction, Km and kcat, were only slightly influenced, but the rate of the hydride transfer reaction was significantly changed by the mutations, as revealed by the deuterium isotope effect on the enzyme activity. These results suggest that site 67 in the flexible loop, being very far from the active site, plays an important role in the stability and function of this enzyme. The characteristics of the mutations were discussed in terms of the modified flexibility of the native structure, compared with the results of mutations at site 121 in another flexible loop.

Base Sequence↗
PubMed1996

Acid and thermal unfolding of Escherichia coli dihydrofolate reductase.

The acid and thermal unfolding of Escherichia coli dihydrofolate reductase (DHFR) were studied by means of circular dichroism (CD) and fluorescence spectroscopy. There existed at least one intermediate around pH 4 in the acid unfolding process at 15 degrees C, in which the tertiary structure was disrupted before unfolding of the secondary structure. The fluorescence energy transfer from intrinsic tryptophan residues to 1-anilinonaphthalene-8-sulfonate suggested the disruption of the tertiary structure around some tryptophan residues of the intermediate. The thermal unfolding process at pH 7.0 also involved at least one intermediate having a disrupted tertiary structure and a folded secondary structure. The three-state thermodynamic analysis showed that the intermediate in thermal unfolding was less stable by 1.8 kcal/mol than the native state. The similarity of the far-ultraviolet CD spectra of acid and thermally unfolded forms suggests that both types of unfolding produce the same structure, which may be a molten globule intermediate such as that in the folding kinetics of DHFR. The acid and thermal unfolding were depressed in the presence of KCl due to stabilization of the native form.

Anilino Naphthalenesulfonates↗
PubMed1995

Compactness of thermally and chemically denatured ribonuclease A as revealed by volume and compressibility.

The conformational changes of ribonuclease A due to thermal and guanidine hydrochloride denaturation were monitored by means of precise density and sound velocity measurements. It was found that the apparent molar volume decreased but the adiabatic compressibility increased on thermal denaturation under acidic conditions (pHs 1.60, 1.90, and 2.08). On the other hand, guanidine hydrochloride denaturation (pH 2.00) brought about large decreases in the compressibility and apparent molar volume. These results indicate that the conformation of the denatured protein is greatly different between the two types of denaturation: the thermally denatured state corresponds to the structure with enhanced thermal fluctuation having a residual secondary structure and a high local concentration of nonpolar groups exposed, but the guanidine hydrochloride denaturation leads to exposure of a large amount of amino acid residues, resulting in an increase in hydration and a decrease in the internal cavity. The compressibility changes due to both types of denaturation were not correlated to a loss of the secondary structure, as judged by means of circular dichroism. These findings suggest that the compactness and thermal fluctuation of the protein cannot be described by a two-state denaturation model and that there are some molten-globule-like intermediates in the denaturation processes.

Animals↗