Surface residues and nonadditive interactions stabilize a consensus homeodomain protein.

Despite the widely reported success of consensus design in producing highly stabilized proteins, little is known about the physical mechanisms underlying this stabilization. Here, we explore the potential sources of stabilization by performing a systematic analysis of the 29 substitutions that we previously found to collectively stabilize a consensus homeodomain compared with an extant homeodomain. By separately introducing groups of consensus substitutions that alter or preserve charge state, occur at varying degrees of residue burial, and occur at positions of varying degrees of conservation, we determine the extent to which these three features contribute to the consensus stability enhancement. Surprisingly, we find that the largest total contribution to stability comes from consensus substitutions on the protein surface and that the largest per substitution contributions come from substitutions that maintain charge state. This finding suggests that, although consensus proteins are often enriched in charged residues, consensus stabilization does not result primarily from interactions involving charged residues. Although consensus substitutions at strongly conserved positions also contribute disproportionately to stabilization, significant stabilization is also contributed from substitutions at weakly conserved positions. Furthermore, we find that identical consensus substitutions show larger stabilizing effects when introduced into the consensus background than when introduced into an extant homeodomain, indicating that synergistic, stabilizing interactions among the consensus residues contribute to consensus stability enhancement of the homeodomain. By measuring DNA binding affinity for the same set of variants, we find that, although consensus design of the homeodomain increases both affinity and folding stability, it does so using a largely nonoverlapping set of substitutions.