A native tertiary interaction stabilizes the A state of cytochrome c.
Certain kinetic intermediates in protein folding are similar to the molten globule, or A state, an equilibrium state of many proteins that is populated under high salt and low pH conditions. Many A states are nearly as compact as native proteins and have native-like secondary structure, but the extent to which nonlocal interactions stabilize the A state is unclear. In this study, thermal denaturation, monitored by circular dichroism, was used to determine the free energy of denaturation of the A state (delta GA<-->D) for Saccharomyces cerevisiae iso-1-ferricytochrome c. Specifically, we examined the wild-type protein, seven variants with amino acid substitutions at the interface between the N- and C-terminal helices, and two variants with mutations at a position close to, but not involved in, the interface. A plot of delta GA<-->D versus delta GN<-->D (the free energy of denaturation of the native state) has a slope near unity, showing that the evolutionarily conserved helix-helix interaction stabilizes the A state to the same degree that it stabilizes the native state.
Marmorino
J L
JL
Department of Chemistry, University of North Carolina, Chapel Hill 27599-3290.
Pielak
G J
GJ
D002942:Circular Dichroism; D003574:Cytochrome c Group / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006358:Hot Temperature; D007700:Kinetics; D017354:Point Mutation; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D013816:Thermodynamics
D003574:Cytochrome c Group