The role of backbone conformational heat capacity in protein stability: Temperature dependent dynamics of the B1 domain of Streptococcal protein G

The contributions of backbone NH group dynamics to the conformational heat capacity of the B1 domain of Streptococcal protein G have been estimated fr om the temperature dependence of N-15 NMR-derived order parameters. Longitu dinal (R-1) and transverse (R-2) relaxation rates, transverse cross-relaxat ion rates (eta(xy)), and steady state {H-1}-N-15 nuclear Overhauser effects were measured at temperatures of 0, 10, 20, 30, 40, and 50 degrees C for 8 9-100% of the backbone secondary amide nitrogen nuclei in the B1 domain. Th e ratio R-2/eta(xy) was used to identify nuclei for which conformational ex change makes a significant contribution to R-2. Relaxation data were fit to the extended model-free dynamics formalism, incorporating an axially symme tric molecular rotational diffusion tensor. The temperature dependence of t he order parameter (S-2) was used to calculate the contribution of each NH group to conformational heat capacity (C-p) and a characteristic temperatur e (T*), representing the density of conformational energy states accessible to each NH group. The heat capacities of the secondary structure regions o f the B1 domain are significantly higher than those of comparable regions o f ether proteins, whereas the heat capacities of less structured regions ar e similar to those in other proteins. The higher local heat capacities are estimated to contribute up to similar to 0.8 kJ/mol K to the total heat cap acity of the B1 domain, without which the denaturation temperature would be similar to 9 degrees C lower (78 degrees C rather than 87 degrees C). Thus , variation of backbone conformational heat capacity of native proteins may be a novel mechanism that contributes to high temperature: stabilization o f proteins.