A NATIVE TERTIARY INTERACTION STABILIZES THE A-STATE OF CYTOCHROME-C

Certain kinetic intermediates in protein folding are similar to the mo lten globule, or A state, an equilibrium state of many proteins that i s populated under high salt and low pH conditions. Many A states are n early as compact as native proteins and have native-like secondary str ucture, but the extent to which nonlocal interactions stabilize the A state is unclear. In this study, thermal denaturation, monitored by ci rcular dichroism, was used to determine the free energy of denaturatio n of the A state (Delta G(A reversible arrow D)) for Saccharomyces cer evisiae iso-1-ferricytochrome c. Specifically, we examined the wild-ty pe protein, seven variants with amino acid substitutions at the interf ace between the N- and C-terminal helices, and two variants with mutat ions at a position close to, but not involved in, the interface. A plo t of Delta G(A reversible arrow D) versus Delta G(N reversible arrow D ) (the free energy of denaturation of the native state) has a slope ne ar unity, showing that the evolutionarily conserved helix-helix intera ction stabilizes the A state to the same degree that it stabilizes the native state.