Core-directed protein design. II. Rescue of a multiply mutated and destabilized variant of ubiquitin

We have applied the method described in the preceding paper [Finucane, M. D ., et al. (1999) Biochemistry 38, 11604-11612], namely, stability-based sel ection using phage display, to explore the sequence requirements for packin g in the hydrophobic core of ubiquitin. In contrast to the parent protein, which was a structurally compromised mutant, the selected variants could be overexpressed and purified in yields for structural studies. In particular , CD and NMR measurements showed that the selectants folded correctly to st able native-like structures. These points demonstrate the utility of our co re-directed method for stabilizing and redesigning proteins. In addition an d in contrast to foregoing studies on other proteins, which suggest that hy drophobic cores permit substitutions provided that hydrophobicity and core volumes are generally conserved, we find that the core of ubiquitin is surp risingly intolerant of amino acid substitutions; variants that survived our selection showed a clear consensus for the wild-type sequence. It is proba ble that our results differed from those from other groups for two reasons. First, ubiquitin may be unusual in that it has strict sequence requirement s for its structure and stability. We discuss this result in light of seque nce conservation in the eukaryotic ubiquitins and proteins of the ubiquitin structural superfamily. Second, our mutants were selected solely on the ba sis of stability, in contrast to the other studies that rely on function-ba sed selection. The latter may lead to proteins that are more plastic and to lerant of substitutions.