We prepared a series of cyt c variants with mutations at these and several other key positions using a novel E. coli expression system that relies on co-expression of the enzyme heme lyase for covalent attachment of the heme group. To simplify the folding behavior, we replaced His33, which is known to form transient non-native interactions with the heme iron during folding, with Asn. Although the order of the two transitions cannot be determined from equilibrium measurements alone, the equilibrium intermediate appears to be closely related to an early kinetic intermediate, based on similarities in the midpoint of the transition and dependence on denaturant concentration. The results indicate that the equilibrium intermediate in the denaturant-induced unfolding transition of the F10I and Y97V variants is compact and contains extensive secondary structure, but lacks the native Met80 heme ligation and specific tertiary interactions characteristic of the native cyt c structure. The unusual unfolding behavior of these mutants indicates that replacement of the specific aromatic ring-ring interaction with a regular hydrophobic contact selectively destabilizes the native structure while allowing accumulation of a partially structured intermediate, which has previously been detected only as a transient kinetic intermediate.
Solid curves represent best fits of a three-state model.
Alternatively, the results can be explained in terms of a model involving a solvent-dependent redistribution of denatured conformations. While formation of an intermediate gives rise to exponential kinetics, the continuous contraction of the polypeptide chain invoked by the latter model would be characterized by a non-exponential time course. In order to discriminate between these possibilities, it is necessary to directly observe the kinetics of folding on the sub-millisecond time scale. These observations were made possible by our recent development of a new continuous-flow capillary mixing apparatus that can resolve fluorescence changes down to the 10 m s range. This suggests that the first kinetic barrier encountered during folding is largely entropic in nature and may represent a general bottleneck caused by an incomplete compensation of unfavorable entropic contributions from favorable intramolecular and solvent interactions.