Structurally engineered cytochromes with unusual ligand-binding properties: Expression of Saccharomyces cerevisiae Met-80 → Ala iso-1-cytochrome c

Yi Lu, Danilo R. Casimiro, Kara L. Bren, John H. Richards, Harry B. Gray

Research output: Contribution to journalArticlepeer-review

Abstract

A strategy has been developed to express and purify a recombinant, nonfunctional axial-ligand mutant of iso-1-cytochrome c (Met-80→ Ala) in Saccharomyces cerevisiae in quantities necessary for extensive biophysical characterization. It involves coexpressing in the same plasmid (YEp213) the nonfunctional gene with a functional gene copy for complementation in a selective medium. The functional gene encodes a product with an engineered metal-chelating dihistidine site (His-39 and Leu-58 → His) that enables efficient separation of the two isoforms by immobilized metal-affinity chromatography. The purified Met-80 → Ala protein possesses a binding site for dioxygen and other exogenous ligands. Absorption spectra of several derivatives of this mutant show striking similarities to those of corresponding derivatives of horse-radish peroxidase, myoglobin, and cytochrome P450. The use of a dual-gene vector for cytochrome c expression together with metal-affinity separation opens the way for the engineering of variants with dramatically altered structural and catalytic properties.

Original languageEnglish (US)
Pages (from-to)11456-11459
Number of pages4
JournalProceedings of the National Academy of Sciences of the United States of America
Volume90
Issue number24
DOIs
StatePublished - Dec 15 1993

Keywords

  • Metal-affinity chromatography
  • Protein engineering
  • Site-directed mutagenesis

ASJC Scopus subject areas

  • General
  • Genetics

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