A Partially Buried Site in Homologous HPr Proteins is Not Optimized for Stability

The energetic consequences of site-specific replacement of a residue at a partially buried site in the two homologous HPr proteins from Escherichia coli and Bacillus subtilis is described. We determined previously that the replacement of a partially buried Lys residue with Glu at position 49 in E. coli HPr increased the conformational stability of the protein substantially because the side-chain of the latter residue could act as a hydrogen-bond acceptor. Here, we extend this analysis to other side-chains with different chemical properties and abilities to form hydrogen bonds to compare the properties of this position in the backgrounds of two different homologous HPr proteins. We find that the variants with polar residues that can form a tertiary hydrogen bond with a nearby site in the protein are more stable than either hydrophobic residues or polar residues that become buried yet are incapable of forming a new hydrogen bond. Furthermore, the protein with the wild-type residue in each HPr variant is not among the most stable of the proteins studied. These results suggest a general strategy for designing variants in which the overall stability of a protein can be modulated in a defined fashion.