title: The significance of denaturant titrations of protein stability: a comparison of rat and baker's yeast cytochrome c and their site-directed asparagine-52-to-isoleucine mutants.
abstract: The residue asparagine-52 of rat cytochrome c and baker's yeast iso-1-cytochrome c was mutated to isoleucine by site-directed mutagenesis, and the unfolding of the wild-type and mutant proteins in urea or guanidinium chloride solutions was studied. Whereas the yeast mutant cytochrome unfolded in 4-7 M urea with a rate constant (k) of 1.7 x 10(-2) s-1, the rat mutant protein unfolded with k = 5.0 x 10(-2) s-1, followed by a slow partial refolding with k = 5.0 x 10(-4) s-1. Denaturant titrations indicated that the mutation increased the stability of the yeast cytochrome by 6.3 kJ (1.5 kcal)/mol, while it decreased that of the rat protein by 11.7 kJ (2.8 kcal)/mol. These results probably reflect structural differences between yeast iso-1 and vertebrate cytochromes c in the vicinity of the Asn-52 side chain.
authors.affiliation: Department of Biological Sciences, University of Illinois at Chicago 60607.
authors.forename: T I
authors.initials: TI
authors.lastname: Koshy
authors.forename: T L
authors.initials: TL
authors.lastname: Luntz
authors.forename: B
authors.initials: B
authors.lastname: Plotkin
authors.forename: A
authors.initials: A
authors.lastname: Schejter
authors.forename: E
authors.initials: E
authors.lastname: Margoliash
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine; D002151:Calorimetry; D003574:Cytochrome c Group / Q000737:chemistry* / Q000378:metabolism; D004355:Drug Stability; D007532:Isoleucine; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D017510:Protein Folding; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D014508:Urea
chemical_list: D003574:Cytochrome c Group; D007532:Isoleucine; D001216:Asparagine; D014508:Urea