Energetics of side chain packing in staphylococcal nuclease assessed by systematic double mutant cycles

All 44 possible double mutant permutations of isoleucine, leucine, and valine were constructed in 11 pairings of six sites in the core of staphylococcal nuclease. The stabilities of these mutants were determined by guanidine hydrochloride denaturation. Comparison of the stabilities of all double mutants with those expected from addition of the corresponding single mutants showed that the effects of the two single mutations are energetically independent of each other in 30 of the double mutants. However, a substantial minority, 14, of the double mutants have stability effects that are not additive. In these cases, it appears that direct van der Waals contacts between the two side chains are present. The requirement of direct van der Waals contact for the interdependence of mutational stability effects is somewhat surprising in light of results previously reported by others. In addition, it was found that double mutants that did not alter or lower the overall number of atoms in the core and that showed nonadditive behavior were more stable than expected from addition of the effects of the corresponding single mutants. A net increase in the number of atoms in the core usually, but not always, resulted in a mutant that was less stable than expected. In contrast to previous staphylococcal nuclease double mutants, energetically significant chan ges to the denatured state do not appear to be occurring in these packing mutants. These conclusions imply that attempts to engineer protein stability based on single mutant data will be generally successful if overall core size is preserved and if residues are not in van der Waals contact.