Exploring the interface between the N- and C-terminal helices of cytochrome c by random mutagenesis within the C-terminal helix.
Buried within cytochrome c lies a highly-conserved helix-helix interface formed by the perpendicular packing of the C-terminal helix against the N-terminal helix. This interface involves a peg-in-hole interaction between Gly-6 and Leu-94 and an aromatic-aromatic interaction between Phe-10 and Tyr-97. To gain insight into protein design, we investigated the relationship between the sequence of the interface and the physiological function of yeast iso-1-cytochrome c. A library of mutants at positions 94 and 97 of the C-terminal helix was created to examine the effect of novel amino acid combinations. We isolated 45 of the 400 possible amino acid combinations, 32 of which result in a functional cytochrome c. Contrary to evolutionary conservation of the peg-in-hole and aromatic-aromatic interactions, we find that side-chain volume and conservation of aromatic residues do not play an essential role in determining function. Additionally, we find negatively-charged residues within the interface that result in a functional cytochrome c. Examination of the 45 missense mutants indicates that approximately 120 unique combinations are compatible with function. These results show that the interface is flexible. However, truncation of the C-terminal helix at position 94 abolishes function, suggesting that the interface is essential. The correlation observed between our library of mutants and the mutation matrix compiled by Gonnet et al. [Gonnet, G. H., Cohen, M. A., & Benner, S. A. (1992) Science 256, 1443-1445] demonstrates the potential use of the matrix to predict the effect of sequence changes on natural proteins and to optimize the design of novel proteins.
Fredericks
Z L
ZL
Department of Chemistry, University of North Carolina, Chapel Hill 27599-3290.
Pielak
G J
GJ
D000595:Amino Acid Sequence; D001482:Base Composition; D001483:Base Sequence; D003062:Codon; D003574:Cytochrome c Group / Q000737:chemistry* / Q000235:genetics; D045304:Cytochromes c; D004252:DNA Mutational Analysis; D015723:Gene Library; D014644:Genetic Variation; D008969:Molecular Sequence Data; D009010:Monte Carlo Method; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins
C014059:CYC1 protein, S cerevisiae; D003062:Codon; D003574:Cytochrome c Group; D029701:Saccharomyces cerevisiae Proteins; D045304:Cytochromes c