REQUIREMENTS FOR PERPENDICULAR HELIX PAIRING

Cassette mutagenesis was used to produce a library of mutations at the interface of the N- and C-terminal helices of Saccharomyces cerevisia e iso-l-cytochrome c. The library is random and comprises >98% mutatio ns, Over 11,000 candidates were assayed for function by selecting for the ability of yeast, with the mutated gene as their sole cytochrome c source, to grow on nonfermentable carbon sources, We estimate that ap proximate to 0.5% of the 160,000 total amino acid combinations at thes e four residues result in a functional cytochrome c, Significant corre lations are found between the phenotype of yeast harboring the alleles and both the Dayhoff mutation matrix and transfer free energies (cycl ohexane-to-water and n-octanol-to-water), Similar correlations are obs erved with respect to growth rate, Finally, sequences that are consist ent with function follow a binary amino acid pattern. (C) 1996 Wiley-L iss, Inc.