NATIVE TERTIARY STRUCTURE IN AN A-STATE

The A-state is an equilibrium species that is thought to represent the molten globule, an on-pathway protein folding intermediate with nativ e secondary structure and non-native, fluctuating tertiary structure. We used yeast iso-l-ferricytochrome c to test for an evolutionary-inva riant tertiary interaction in its A-state. Thermal denaturation monito red by circular dichroism (CD)spectropolarimetry was used to determine A-state and native-state stabilities, Delta G(A reversible arrow D) a nd Delta G(N reversible arrow D). We examined the wild-type protein, s even variants with substitutions at the interface between the N and C- terminal helices, and four control variants. The controls have the sam e amino acid changes as the interface variants, but the changes are cl ose to, not at, the interface. We also examined the pH and sulfate con centration dependencies and found that while these factors affect the far-UV CD spectra of the least stable variants, they do not alter the difference in stability between the wild-type protein and the variants . A Delta G(A reversible arrow D) versus-Delta G(N reversible arrow D) plot for the interface variants has a slope near unity and the contro l variants have near-wild-type stability. These results show that the helix-helix interaction stabilizes the A-state and the native state to the same degree, confirming our preliminary report. We determined tha t the heat capacity change for A-state denaturation is approximate to 60% of the value for native-state denaturation, indicating that the A- state interior is native-like. We discuss our results in relation to f erricytochrome c folding kinetics. (C) 1998 Academic Press Limited.