Tadashi Kamiyama

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.

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.

Evolutional Design of a Hyperactive Cysteine- and Methionine-free Mutant of Escherichia coli Dihydrofolate Reductase

We developed a strategy for finding out the adapted variants of enzymes, and we applied it to an enzyme, dihydrofolate reductase (DHFR), in terms of its catalytic activity so that we successfully obtained several hyperactive cysteine- and methionine-free variants of DHFR in which all five methionyl and two cysteinyl residues were replaced by other amino acid residues. Among them, a variant (M1A/M16N/M20L/M42Y/C85A/M92F/C152S), named as ANLYF, has an approximately seven times higher kcat value than wild type DHFR. Enzyme kinetics and crystal structures of the variant were investigated for elucidating the mechanism of the hyperactivity. Steady-state and transient binding kinetics of the variant indicated that the kinetic scheme of the catalytic cycle of ANLYF was essentially the same as that of wild type, showing that the hyperactivity was brought about by an increase of the dissociation rate constants of tetrahydrofolate from the enzyme-NADPH-tetrahydrofolate ternary complex. The crystal structure of the variant, solved and refined to an R factor of 0.205 at 1.9-Å resolution, indicated that an increased structural flexibility of the variant and an increased size of the N-(p-aminobenzoyl)-l-glutamate binding cleft induced the increase of the dissociation constant. This was consistent with a large compressibility (volume fluctuation) of the variant. A comparison of folding kinetics between wild type and the variant showed that the folding of these two enzymes was similar to each other, suggesting that the activity enhancement of the enzyme can be attained without drastic changes of the folding mechanism.

Effects of mutation and ligand binding on the compressibility of a protein

The partial specific volume ( v o ) and adiabatic compressibility ( βs o ) of E. coli dihydrofolate reductase (DHFR) are largely influenced by binding ligands (DHF, THF, NADPH, NADP + , and methotrexate) and single amino acid substitution at Gly67, Gly121, and Ala145 located in three flexible loops (18 mutants). The variations in v o and β s o are mainly ascribed to the amount of cavity with a minor contribution of hydration (solvent accessible surface area). The β s o of DHFR changes alternatively by binding or releasing the coenzyme and substrate. A mutant with a large β s o value shows high enzymatic activity. These results demonstrate that a small alteration in the local structure due to ligand binding and mutation is dramatically magnified in the overall protein dynamics to affect the function, possibly via modification of cavity or long range interactions.