title: Cytochrome c folds through foldon-dependent native-like intermediates in an ordered pathway.
abstract: Previous hydrogen exchange (HX) studies of the spontaneous reversible unfolding of Cytochrome c (Cyt c) under native conditions have led to the following conclusions. Native Cyt c (104 residues) is composed of five cooperative folding units, called foldons. The high-energy landscape is dominated by an energy ladder of partially folded forms that differ from each other by one cooperative foldon unit. The reversible equilibrium unfolding of native Cyt c steps up through these intermediate forms to the unfolded state in an energy-ordered sequence, one foldon unit at a time. To more directly study Cyt c intermediates and pathways during normal energetically downhill kinetic folding, the present work used HX pulse labeling analyzed by a fragment separation-mass spectrometry method. The results show that 95% or more of the Cyt c population folds by stepping down through the same set of foldon-dependent pathway intermediates as in energetically uphill equilibrium unfolding. These results add to growing evidence that proteins fold through a classical pathway sequence of native-like intermediates rather than through a vast number of undefinable intermediates and pathways. The present results also emphasize the condition-dependent nature of kinetic barriers, which, with less informative experimental methods (fluorescence, etc.), are often confused with variability in intermediates and pathways.
authors.affiliation: Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104.
authors.forename: Wenbing
authors.initials: W
authors.lastname: Hu
authors.affiliation: Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104.
authors.forename: Zhong-Yuan
authors.initials: ZY
authors.lastname: Kan
authors.affiliation: Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104.
authors.forename: Leland
authors.initials: L
authors.lastname: Mayne
authors.affiliation: Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104 engl@mail.med.upenn.edu.
authors.forename: S Walter
authors.initials: SW
authors.lastname: Englander
mesh_terms: D045304:Cytochromes c / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D017510:Protein Folding; D013816:Thermodynamics
chemical_list: D045304:Cytochromes c
keywords: cytochrome c; foldons; protein folding