Tolerance of a protein to multiple polar-to-hydrophobic surface substitutions

Hydrophobic substitutions at solvent-exposed positions in two a-helical reg ions of the bacteriophage P22 Are repressor were introduced by combinatoria l mutagenesis. In helix A, hydrophobic residues were tolerated individually at each of the five positions examined, but multiple substitutions were po orly tolerated as shown by the finding that mutants with more than two addi tional hydrophobic residues were biologically inactive. Several inactive he lix A variants were purified and found to have reduced thermal stability re lative to wild-type Are, with a rough correlation between the number of pol ar-to-hydrophobic substitutions and the magnitude of the stability defect. Quite different results were obtained in helix B, where variants with as ma ny as five polar-to-hydrophobic substitutions were found to be biologically active and one variant with three hydrophobic substitutions had a t(m) 6 d egrees C higher than wild-type. By contrast, a helix A mutant with three si milar polar-to-hydrophobic substitutions was 23 degrees C less stable than wild-type. Also, one set of three polar-to-hydrophobic substitutions in hel ix B was tolerated when introduced into the wild-type background but not wh en introduced into an equally active mutant having a nearly identical struc ture. Context effects occur both when comparing different regions of the sa me protein and when comparing the same region in two different homologues.