Title: Coupled kinetic traps in cytochrome c folding: His-Heme misligation and proline isomerization
Abstract: The effect of His-heme misligation on folding has been investigated for a triple mutant of yeast iso-2 cytochrome c (N26H,H33N,H39K iso-2). The variant contains a single misligating His residue at position 26, a location at which His residues are found in several cytochrome c homologues, including horse, tuna, and yeast iso-1. The amplitude for fast phase folding exhibits a strong initial pH dependence. For GdnHCl unfolded protein at an initial pH < 5, the observed refolding at final pH 6 is dominated by a fast phase (τ2f = 20 ms, α2f = 90 %) that represents folding in the absence of misligation. For unfolded protein at initial pH 6, folding at final pH 6 occurs in a fast phase of reduced amplitude (α2f ∼ 20 %) but the same rate (τ2f = 20 ms), and in two slower phases (τm = 6-8 seconds, αm ∼ 45 %; and τ1b = 16-20 seconds, α1b ∼ 35 %). Double jump experiments show that the initial pH dependence of the folding amplitudes results from a slow pH-dependent equilibrium between fast and slow folding species present in the unfolded protein. The slow equilibrium arises from coupling of the His protonation equilibrium to His-heme misligation and proline isomerization. Specifically, Pro25 is predominantly in trans in the unligated low-pH unfolded protein, but is constrained in a non-native cis isomerization state by His26-heme misligation near neutral pH. Refolding from the misligated unfolded form proceeds slowly due to the large energetic barrier required for proline isomerization and displacement of the misligated His26-heme ligand.
Publication Year: 2000
Publication Date: 2000-05-01
Language: en
Type: article
Indexed In: ['crossref', 'pubmed']
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Cited By Count: 32
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