Construction and Characterization of Mutant Iso-2-cytochromes c with Replacement of Conserved Prolines

Ladonna C. Wood, Kamalam Muthukrishnan, Terry B. White, Latha Ramdas, Barry T. Nall

Research output: Contribution to journalArticlepeer-review

23 Scopus citations

Abstract

Oligonucleotide-directed mutagenesis has been used to construct two mutant forms of iso-2-cytochrome c. In one, Pro-30 is replaced by threonine; in the other, Pro-76 is replaced by glycine. Both prolines are fully conserved among mitochondrial cytochromes c and play important structural and functional roles. Yeast with either the Pro-30 or the Gly-76 mutation has appreciable levels of mutant protein in vivo and grows on media containing nonfermentable carbon sources. Thus, neither mutation blocks protein targeting to mitochondria, uptake by mitochondria, covalent attachment of heme, or in vivo function. As judged by ultraviolet-visible spectrophotometry and proton nuclear magnetic resonance spectroscopy, the nativelike conformation of purified Gly-76 iso-2 at pH 6 is almost indistinguishable from that of the normal protein at pH 6. Ultraviolet second-derivative spectrophotometry, however, suggests an increase in the average number of exposed tyrosine side chains, with 2.25 out of 5 residues exposed for the mutant compared to 1.95 for normal iso-2. Above neutral pH, the protein folds to a mutant conformation possibly related to alkaline cytochrome c. Nuclear Overhauser difference spectroscopy of the reduced nativelike conformation allows assignment of several proton resonances and comparison of side-chain conformations of the heme ligand Met-80 in the mutant and the normal proteins. The proton chemical shifts for the assigned resonances are the same within errors for Gly-76 iso-2 and normal iso-2 at pD 6, 20 °C. A distorted resonance line shape and small changes in the nuclear Overhauser difference spectrum, however, suggest a minor change in conformation of the mutant protein involving the β–H of the Met-80 side chain.

Original languageEnglish (US)
Pages (from-to)8554-8561
Number of pages8
JournalBiochemistry
Volume27
Issue number23
DOIs
StatePublished - Nov 1 1988

ASJC Scopus subject areas

  • Biochemistry

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