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 cerevisiae iso-1-cytochrome c. The library is random and comprises > 98% mutations. 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 approximately 0.5% of the 160,000 total amino acid combinations at these four residues result in a functional cytochrome c. Significant correlations are found between the phenotype of yeast harboring the alleles and both the Dayhoff mutation matrix and transfer free energies (cyclohexane-to-water and n-octanol-to-water). Similar correlations are observed with respect to growth rate. Finally, sequences that are consistent with function follow a binary amino acid pattern.
Beasley
J R
JR
Department of Chemistry, University of North Carolina at Chapel Hill 27599-3290, USA.
Pielak
G J
GJ
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics; D001483:Base Sequence; D002455:Cell Division / Q000235:genetics; D003001:Cloning, Molecular; D003574:Cytochrome c Group / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D015723:Gene Library; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000235:genetics; D009154:Mutation / Q000235:genetics; D010641:Phenotype; D010957:Plasmids / Q000235:genetics; D017433:Protein Structure, Secondary; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000235:genetics
D000596:Amino Acids; D003574:Cytochrome c Group; D007527:Isoenzymes