Partially formed native tertiary interactions in the A-state of cytochromec

Considerable insight into protein structure, stability, and folding has bee n obtained from studies of non-native states. We have studied the extent of native tertiary contacts in one such molecule, the A-state of yeast iso-1- ferricytochrome c. Previously, we showed that the interface between the N a nd C-terminal helices is completely formed in the A-state. Here, we focus o n interactions essential for forming the heme pocket of eukaryotic cytochro mes c. To determine the extent of these interactions, we used saturation mu tagenesis at the evolutionarily invariant residue leucine 68, and measured the free energy of denaturation for the native states and the A-states of f unctional variants. We show that, unlike the interaction between the termin al helices, the native interactions between the 60s helix and the rest of t he protein are not completely formed in the A-state. (C) 1999 Academic Pres s.