Denatured states of yeast cytochrome c induced by heat and guanidinium chloride are structurally and thermodynamically different

We have been interested in understanding the question: What is the role of these five extra N-terminal residues in folding and stability of the protein? To answer this question we have prepared five deletants of y-cyt- c by sequentially removing these extra residues.

During our studies on the wild type (WT) protein and its deletants, we observed that the amount of secondary structure in the guanidinium chloride (GdmCl)-induced denatured (D) state of each protein is different from that of the heat-induced denatured (H) state. This finding is confirmed by the observation of an additional cooperative transition curve of optical properties between H and D states on the addition of different concentrations of GdmCl to the already heat denatured WT y-cyt- c and its deletants at pH 6.0 and 68°C. For each protein, analysis of transition curves representing processes, native (N) state ↔ D state, N state ↔ H state, and H state ↔ D state, was done to obtain Gibbs free energy changes associated with all the three processes.