Multiple mutations at distally located sites have been introduced into yeast iso-1 cytochrome c to determine the contributions of three amino acids to the structural and functional properties of this protein. The mutant proteins, for which high-resolution structures were determined, included all possible combinations of the substitutions Arg38Ala, Asn52Ile, and Phe82Ser. Arg38, Asn52, and Phe82 are all conserved in the primary sequences of eukaryotic cytochromes c and have been shown to significantly affect several properties of these proteins including protein stability, heme reduction potential, and oxidation state dependent conformational changes. The present studies show that the structural consequences of each amino acid substitution in combinatorial mutant proteins were similar to those observed in the related single-mutant proteins, and therefore no synergistic effect between mutation sites was observed for this feature. With respect to protein stability, the effect of individual mutations can be understood from the structural changes observed for each. It is found that stability effects of the three mutation sites are independent and cumulative in multiple-mutant proteins. This reflects the independent nature of the structural changes induced at the three distally located mutation sites. In terms of heme reduction potential two effects are observed. For substitution of Phe82 by serine, the mechanism by which reduction potential is lowered is different from that occurring at either the Arg38 or the Asn52 site and is independent of residue replacements at these latter two positions. For Arg38 and Asn52, overlapping interactions lead to a higher reduction potential than expected from a strict additive effect of substitutions at these residues. This appears to arise from interaction of these two amino acids with a common heme element, namely, the heme propionate A group. The present results underscore the difficulty of predicting synergistic effects of multiple mutations within a protein.

• PMID: 7711047
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Journal Article | Research Support, Non-U.S. Gov't | Research Support, U.S. Gov't, Non-P.H.S. | Research Support, U.S. Gov't, P.H.S.

Authors

Lo TP, Komar-Panicucci S, Sherman F, McLendon G, Brayer GD

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