An important goal of protein design is to understand the forces that stabilize a particular fold in preference to alternative folds. Here, we describe an extension of earlier studies in which we successfully designed a stable, native-like helical protein that is 50% identical in sequence to a predominantly β-sheet protein, the B1 domain of Streptococcal IgG-binding protein G. We report the characteristics of a series of variants of our original design that have even higher sequence identity to the B1 domain. Their properties illustrate the extent to which protein stability and conformation can be modulated through careful manipulation of key amino acid residues. Our results have implications for understanding conformational change phenomena of central biological importance and in probing the malleability of the sequence/structure relationship.

The contributions of backbone NH group dynamics to the conformational heat capacity of the B1 domain of Streptococcal protein G have been estimated from the temperature dependence of 15N NMR-derived order parameters. Longitudinal (R1) and transverse (R2) relaxation rates, transverse cross-relaxation rates (ηxy), and steady state {1H}–15N nuclear Overhauser effects were measured at temperatures of 0, 10, 20, 30, 40, and 50 °C for 89–100% of the backbone secondary amide nitrogen nuclei in the B1 domain. The ratio R2/ηxy was used to identify nuclei for which conformational exchange makes a significant contribution to R2. Relaxation data were fit to the extended model-free dynamics formalism, incorporating an axially symmetric molecular rotational diffusion tensor. The temperature dependence of the order parameter (S2) was used to calculate the contribution of each NH group to conformational heat capacity (Cp) and a characteristic temperature (T*), representing the density of conformational energy states accessible to each NH group. The heat capacities of the secondary structure regions of the B1 domain are significantly higher than those of comparable regions of other proteins, whereas the heat capacities of less structured regions are similar to those in other proteins. The higher local heat capacities are estimated to contribute up to ∼0.8 kJ/mol K to the total heat capacity of the B1 domain, without which the denaturation temperature would be ∼9 °C lower (78 °C rather than 87 °C). Thus, variation of backbone conformational heat capacity of native proteins may be a novel mechanism that contributes to high temperature stabilization of proteins.