title: Constraints on amino acid substitutions in the N-terminal helix of cytochrome c explored by random mutagenesis.
abstract: The interaction of the N- and C-terminal helices is a hallmark of the cytochrome c family. Oligodeoxyribonucleotide-directed random mutagenesis within the gene encoding the C102T protein variant of Saccharomyces cerevisiae iso-1-cytochrome c was used to generate a library of mutations at the evolutionary invariant residues Gly-6 and Phe-10 in the N-terminal helix. Transformation of this library (contained on a low-copy-number yeast shuttle phagemid) into a yeast strain lacking a functional cytochrome c, followed by selection for cytochrome c function, reveals that 4-10% of the 400 possible amino acid substitutions are compatible with function. DNA sequence analysis of phagemids isolated from transformants exhibiting the functional phenotype elucidates the requirements for a stable helical interface. Basic residues are not tolerated at position 6 or 10. There is a broad volume constraint for amino acids at position 6. The amino acid substitutions observed to be compatible with function at Phe-10 show that the hydrophobic effect alone is sufficient to promote helical association. There are severe constraints that limit the combinations consistent with function, but the number of functionally consistent combinations observed exemplifies the plasticity of proteins.
authors.affiliation: Department of Chemistry, University of North Carolina, Chapel Hill 27599-3290.
authors.forename: D S
authors.initials: DS
authors.lastname: Auld
authors.forename: G J
authors.initials: GJ
authors.lastname: Pielak
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics*; D001483:Base Sequence; D003574:Cytochrome c Group / Q000235:genetics*; D015698:Genomic Library; D005998:Glycine / Q000235:genetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010641:Phenotype; D010649:Phenylalanine / Q000235:genetics; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000235:genetics
chemical_list: D000596:Amino Acids; D003574:Cytochrome c Group; D010649:Phenylalanine; D005998:Glycine