title: Analysis of the structure and stability of omega loop A replacements in yeast iso-1-cytochrome c.
abstract: Omega (omega)-loop A, residues 18-32 in wild-type yeast iso-1-cytochrome c, has been deleted and replaced with loop sequences from three other cytochromes c and one from esterase. Yeast expressing a partial loop deletion do not contain perceptible amounts of holoprotein as measured by low-temperature spectroscopy and cannot grow on nonfermentable media. Strains expressing loop replacement mutations accumulate holoprotein in vivo, but the protein function varies depending on the sequence and length of the replacement loop; in vivo expression levels do not correlate with their thermal denaturation temperatures. In vitro spectroscopic studies of the loop replacement proteins indicate that all fold into a native-like cytochrome c conformation, but are less stable than the wild-type protein. Decreases in thermal stability are caused by perturbation of loop C backbone in one case and a slight reorganization of the protein hydrophobic core in another case, rather than rearrangement of the loop A backbone. A single-site mutation in one of the replacement mutants designed to relieve inefficient hydrophobic core packing caused by the new loop recovers some, but not all, of the lost stability.
authors.affiliation: Department of Biological Sciences, University at Albany, SUNY 12222, USA. jacque@isadora.albany.edu
authors.forename: J S
authors.initials: JS
authors.lastname: Fetrow
authors.forename: S R
authors.initials: SR
authors.lastname: Horner
authors.forename: W
authors.initials: W
authors.lastname: Oehrl
authors.forename: D L
authors.initials: DL
authors.lastname: Schaak
authors.forename: T L
authors.initials: TL
authors.lastname: Boose
authors.forename: R E
authors.initials: RE
authors.lastname: Burton
mesh_terms: D000595:Amino Acid Sequence; D002942:Circular Dichroism; D003574:Cytochrome c Group / Q000737:chemistry*; D045304:Cytochromes c; D004795:Enzyme Stability; D006358:Hot Temperature; D008969:Molecular Sequence Data; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins; D013056:Spectrophotometry, Ultraviolet
chemical_list: C014059:CYC1 protein, S cerevisiae; D003574:Cytochrome c Group; D029701:Saccharomyces cerevisiae Proteins; D045304:Cytochromes c