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 selection using phage display, to explore the sequence requirements for packing 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 stable native-like structures. These points demonstrate the utility of our core-directed method for stabilizing and redesigning proteins. In addition and in contrast to foregoing studies on other proteins, which suggest that hydrophobic cores permit substitutions provided that hydrophobicity and core volumes are generally conserved, we find that the core of ubiquitin is surprisingly intolerant of amino acid substitutions; variants that survived our selection showed a clear consensus for the wild-type sequence. It is probable that our results differed from those from other groups for two reasons. First, ubiquitin may be unusual in that it has strict sequence requirements for its structure and stability. We discuss this result in light of sequence conservation in the eukaryotic ubiquitins and proteins of the ubiquitin structural superfamily. Second, our mutants were selected solely on the basis of stability, in contrast to the other studies that rely on function-based selection. The latter may lead to proteins that are more plastic and tolerant of substitutions.
Finucane
M D
MD
Centre for Biomolecular Design, School of Biological Sciences, University of Sussex, Falmer, Brighton BN1 9QG, U.K.
Woolfson
D N
DN
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002942:Circular Dichroism; D017931:DNA Primers; D006358:Hot Temperature; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D014452:Ubiquitins / Q000737:chemistry* / Q000235:genetics
D017931:DNA Primers; D014452:Ubiquitins