Solvent isotope effect on the differences in structure and stability between normal and deuterated proteins

Differential scanning microcalorimetry was used to investigate the enthalpy (ΔH_d) and the temperature (t_d) of thermal denaturation of normal (nondeuterated) (H-PC) and deuterated (D-PC) phycocyanins in D₂O solvent. Values of t_d in D-PC are about 5–7°C lower than those in H-PC. The magnitudes of ΔH_d in D-PC are only 21–32% of those in H-PC. During the protein unfolding, the heat-capacity changes (ΔC_p) in D-PC are also lower than those in H-PC. CD was employed to evaluate the secondary structure and the urea denaturation of these proteins in D₂O solvent. These proteins have about the same α-helix content. D-PC is less resistant to the denaturant urea than is H-PC. In general, the apparent free-energy change in the process of protein unfolding at zero denaturant concentration is higher in H-PC than in D-PC. Comparisons of the present results for D₂O solvent with those previously reported for H₂O reveal that solvent isotope effect essentially does not change the α-helix content in H-PC and D-PC. However, D-PC or H-PC has a higher random-coil content in its secondary structure in D₂O than in H₂O. Substitution of H₂O with D₂O as the solvent increases t_d in both D-PC and H-PC, lowers ΔH_d in H-PC, and greatly lowers ΔH_d in D-PC. The deuterium solvent isotope effect does not change ΔC_p in H-PC but lowers ΔC_p in D-PC. In the urea denaturation, the magnitudes of (C_u)_1/2 in H-PC and D-PC are not affected by such a solvent effect, whereas those of ΔG are greatly increased. These results are correlated with the structure and stability of the proteins.