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

All 44 possible double mutant permutations of isoleucine, leucine, and vali ne were constructed in 11 pairings of six sites in the core of staphylococc al nuclease. The stabilities of these mutants were determined by guanidine hydrochloride denaturation. Comparison of the stabilities of all double mut ants with those expected from addition of the corresponding single mutants showed that the effects of the two single mutations are energetically indep endent of each other in 30 of the double mutants. However, a substantial mi nority, 14, of the double mutants have stability effects that are not addit ive. 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 co ntact for the interdependence of mutational stability effects is somewhat s urprising in light of results previously reported by others. In addition, i t was found that double mutants that did not alter or lower the overall num ber of atoms in the core and that showed nonadditive behavior were more sta ble 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 contra st to previous staphylococcal nuclease double mutants, energetically signif icant chan ges to the denatured state do not appear to be occurring in thes e packing mutants. These conclusions imply that attempts to engineer protei n stability based on single mutant data will be generally successful if ove rall core size is preserved and if residues are not in van der Waals contac t.