title: Changes in conformation and slow refolding kinetics in mutant iso-2-cytochrome c with replacement of a conserved proline residue.
abstract: Using oligonucleotide-directed mutagenesis, we have produced a mutant form of iso-2-cytochrome c of yeast in which threonine (Thr-71) replaces a conserved proline residue (Pro-71) located between two short alpha-helical segments in the native protein. Optical spectroscopy indicates that, at pH 7.2, Thr-71 iso-2-cytochrome c folds to a nonnative conformation possibly related to the alkaline form of the native protein. On titration to pH 5.2, Thr-71 iso-2-cytochrome c regains many of the optical properties of the normal protein. We have shown that the proline residue at position 71 has no effect on the kinetics of fluorescence-detected slow refolding. However, between pH 5 and pH 7.2 the amplitude for absorbance-detected slow folding is strongly pH dependent in the mutant protein but is largely independent of pH in the normal protein. We believe this to be due to the folding of Thr-71 iso-2-cytochrome c to a nonnative conformation at pH 7.2 that does not require the slow, absorbance-detected conformational changes observed in folding to the more native-like state at pH 5-6.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Texas Medical School at Houston 77225.
authors.forename: T B
authors.initials: TB
authors.lastname: White
authors.forename: P B
authors.initials: PB
authors.lastname: Berget
authors.forename: B T
authors.initials: BT
authors.lastname: Nall
mesh_terms: D000595:Amino Acid Sequence; D003574:Cytochrome c Group / Q000235:genetics* / Q000378:metabolism; D045304:Cytochromes c; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005822:Genetic Vectors; D009154:Mutation; D011392:Proline; D011487:Protein Conformation; D011489:Protein Denaturation; D013053:Spectrophotometry
chemical_list: D003574:Cytochrome c Group; C032139:iso-2-cytochrome C; D045304:Cytochromes c; D011392:Proline