Effect of an Imposed Contact on Secondary Structure in the Denatured State of Yeast Iso-1-cytochrome c.

There is considerable evidence that long-range interactions stabilize residual protein structure under denaturing conditions. However, evaluation of the effect of a specific contact on structure in the denatured state has been difficult. Iso-1-cytochrome c variants with a Lys54 → His mutation form a particularly stable His-heme loop in the denatured state, suggestive of loop-induced residual structure. We have used multidimensional nuclear magnetic resonance methods to assign 1 H and 15 N backbone amide and 13 C backbone and side chain chemical shifts in the denatured state of iso-1-cytochrome c carrying the Lys54 → His mutation in 3 and 6 M guanidine hydrochloride and at both pH 6.4, where the His54-heme loop is formed, and pH 3.6, where the His54-heme loop is broken. Using the secondary structure propensity score, with the 6 M guanidine hydrochloride chemical shift data as a random coil reference state for data collected in 3 M guanidine hydrochloride, we found residual helical structure in the denatured state for the 60s helix and the C-terminal helix, but not in the N-terminal helix in the presence or absence of the His54-heme loop. Non-native helical structure is observed in two regions that form Ω-loops in the native state. There is more residual helical structure in the C-terminal helix at pH 6.4 when the loop is formed. Loop formation also appears to stabilize helical structure near His54, consistent with induction of helical structure observed when His-heme bonds form in heme-peptide model systems. The results are discussed in the context of the folding mechanism of cytochrome c.