Intimate View of a Kinetic Protein Folding Intermediate: Residue-resolved Structure, Interactions, Stability, Folding and Unfolding Rates, Homogeneity

Krishna, M.M.G.; Lin, Y.; Mayne, L.; Englander, S.Walter.

Journal of Molecular Biology 334(3): 501-513


ISSN/ISBN: 0022-2836
PMID: 14623190
DOI: 10.1016/j.jmb.2003.09.070
Accession: 009888394

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A cytochrome c kinetic folding intermediate was studied by hydrogen exchange (HX) pulse labeling. Advances in the technique and analysis made it possible to define the structured and unstructured regions, equilibrium stability, and kinetic opening and closing rates, all at an amino acid-resolved level. The entire N-terminal and C-terminal helices are formed and docked together at their normal native positions. They fray in both directions from the interaction region, due to a progression in both unfolding and refolding rates, leading to the surprising suggestion that helix propagation may proceed very slowly in the condensed milieu. Several native-like beta turns are formed. Some residues in the segment that will form the native 60s helix are protected but others are not, suggesting energy minimization to some locally non-native conformation in the transient intermediate. All other regions are unprotected, presumably dynamically disordered. The intermediate resembles a partially constructed native state. It is early, on-pathway, and all of the refolding molecules pass through it. These and related results consistently point to distinct, homogeneous, native-like intermediates in a stepwise sequential pathway, guided by the same factors that determine the native structure. Previous pulse labeling efforts have always assumed EX2 exchange during the labeling pulse, often leading to the suggestion of heterogeneous intermediates in alternative parallel pathways. The present work reveals a dominant role for EX1 exchange in the high pH labeling pulse, which will mimic heterogeneous behavior when EX2 exchange is assumed. The general problem of homogeneous versus heterogeneous intermediates and pathways is discussed. Reprinted by permission of the publisher.