Unfolding cytochromes c-b₅₆₂ and Rd apo b₅₆₂

Abstract

We have analyzed the early stages of unfolding of cytochromes c-b₅₆₂ (PDB ID: 2BC5) and Rd apo b₅₆₂ (PDB ID: 1YYJ). Our geometrical approach proceeds from an analysis of the crystal structure reported for each protein. We quantify, residue-by-residue and region-by-region, the spatial and angular changes in the structure as the protein denatures, and quantify differences that result from the seven residues that differ in the two proteins. Using two independent analyses, one based on spatial metrics and the second on angular metrics, we establish the order of unfolding of the five helices in cyt c-b₅₆₂ and the four helices in the apo protein. For the two helices nearest the N-terminal end of both proteins, the ones in the apo protein unfold first. For the two helices nearest the C-terminal end, the interior helix of the apo protein unfolds first, whereas the terminal helix of the holo protein unfolds first. Excluded-volume effects (repulsive interactions) are minimized in turning regions; the overall range in Δ values is Δ = 36.3 ų for cyt c-b₅₆₂ and Δ = 36.6 ų for the apo protein, whereas the span for all 20 amino acids is Δ = 167.7 ų. As our work indicates that the interior helix of cytochrome c-b₅₆₂ is the first to fold, we suggest that this helix protects the heme from misligation, consistent with ultrafast folding over a minimally frustrated funneled landscape.

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