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  • Title: Analysis of the invariant Phe82 residue of yeast iso-1-cytochrome c by site-directed mutagenesis using a phagemid yeast shuttle vector.
    Author: Inglis SC, Guillemette JG, Johnson JA, Smith M.
    Journal: Protein Eng; 1991 Jun; 4(5):569-74. PubMed ID: 1653956.
    Abstract:
    A phagemid (pING4) carrying the yeast iso-1-cytochrome c gene was constructed which bears all the elements necessary for replication in yeast and bacteria and may be converted into a single-stranded form of DNA for site-directed mutagenesis and nucleotide sequencing. The recombinant vector was used to create a complete set of 19 amino acid changes at position 82, a phylogenetically conserved phenylalanine residue in mitochondrial cytochrome c. All the different forms of cytochrome c were functional in vivo, based upon their ability to support respiration when the mutant proteins were expressed in a yeast strain (otherwise devoid of cytochrome c) grown on non-fermentable carbon sources, with only the strain containing the Cys82 variant having a substantially decreased growth rate. These results are interpreted in terms of the available structural and functional information previously reported on a subset of cytochrome c proteins with mutations at position 82.
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