THE CRYSTAL-STRUCTURE OF A MUTANT PROTEIN WITH ALTERED BUT IMPROVED HYDROPHOBIC CORE PACKING

The dense packing observed in protein interiors appears to be crucial for stabilizing the native structure-even subtle internal substitution s are usually destabilizing. Thus, steric complementarity of core resi dues is thought to be an important criterion for ''inverse folding'' p redictive methods, which judge whether a newly determined sequence is consistent with any known folds. A major problem in the development of useful core packing evaluation algorithms, however, is that there are occasional mutations that are predicted to disrupt native packing but that yield an equally or more stable protein. We have solved the crys tal structure of such a variant of A repressor, which, despite having three larger core substitutions, is more stable than the wild type. Th e structure reveals that the protein accommodates the potentially disr uptive residues with shifts in its alpha-helical arrangement. The vari ant is apparently more stable because its packing is improved-the core has a higher packing density and little geometric strain. These rearr angements, however, cause repositioning of functional residues, which result in reduced DNA binding activity. By comparing these results wit h the predictions of two core packing algorithms, it is clear that the protein possesses a relatively high degree of main-chain flexibility that must be accounted for in order to predict the full spectrum of co mpatible core sequences. This study also shows how, in protein evoluti on, a particular set of core residue identities might be selected not because they provide optimal stability but because they provide suffic ient stability in addition to the precise structure required for optim al activity.