Energetics of side chain packing in staphylococcal nuclease assessed by exchange of valines, isoleucines, and leucines

To examine the importance of side chain packing to protein stability, each of the I I leucines in staphylococcal nuclease was substituted with isoleuc ine and valine. The nine valines were substituted with leucine and isoleuci ne, while the five isoleucines, previously substituted with valine, were su bstituted with leucine and methionine. These substitutions conserve the hyd rophobic character of these side chains but alter side chain geometry and, in some cases, size. In addition, eight threonine residues, previously subs tituted with valine, were substituted with isoleucine to test the importanc e of packing at sites normally not occupied by a hydrophobic residue. The s tabilities of these 58 mutant proteins were measured by guanidine hydrochlo ride denaturation. To the best of our knowledge, this is the largest librar y of single packing mutants yet characterized. As expected, repacking stabi lity effects are tied to the degree of side chain burial. The average energ etic cost of moving a single buried methyl group was 0.9 kcal/mol, albeit w ith a standard deviation of 0.8 kcal/mol. This average is actually slightly greater than the value of 0.7-0.8 kcal/mol estimated for the hydrophobic t ransfer energy of a methylene from octanol to water. These results appear t o indicate that van der Waals interactions gained from optimal packing are at least as important in stabilizing the native state of proteins as hydrop hobic transfer effects.