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.
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.
Koshy
T I
TI
Department of Biological Sciences, University of Illinois at Chicago 60607.
Luntz
T L
TL
Plotkin
B
B
Schejter
A
A
Margoliash
E
E
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
D003574:Cytochrome c Group; D007532:Isoleucine; D001216:Asparagine; D014508:Urea