title: Ultrafast hydrogen exchange reveals specific structural events during the initial stages of folding of cytochrome c.
abstract: Many proteins undergo a sharp decrease in chain dimensions during early stages of folding, prior to the rate-limiting step in folding. However, it remains unclear whether compact states are the result of specific folding events or a general hydrophobic collapse of the poly peptide chain driven by the change in solvent conditions. To address this fundamental question, we extended the temporal resolution of NMR-detected H/D exchange labeling experiments into the microsecond regime by adopting a microfluidics approach. By observing the competition between H/D exchange and folding as a function of labeling pH, coupled with direct measurement of exchange rates in the unfolded state, we were able to monitor hydrogen-bond formation for over 50 individual backbone NH groups within the initial 140 microseconds of folding of horse cytochrome c. Clusters of solvent-shielded amide protons were observed in two α-helical segments in the C-terminal half of the protein, while the N-terminal helix remained largely unstructured, suggesting that proximity in the primary structure is a major factor in promoting helix formation and association at early stages of folding, while the entropically more costly long-range contacts between the N- and C-terminal helices are established only during later stages. Our findings clearly indicate that the initial chain condensation in cytochrome c is driven by specific interactions among a subset of α-helical segments rather than a general hydrophobic collapse.
authors.affiliation: Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, United States.
authors.forename: Hossein
authors.initials: H
authors.lastname: Fazelinia
authors.forename: Ming
authors.initials: M
authors.lastname: Xu
authors.forename: Hong
authors.initials: H
authors.lastname: Cheng
authors.forename: Heinrich
authors.initials: H
authors.lastname: Roder
mesh_terms: D045304:Cytochromes c / Q000737:chemistry* / Q000302:isolation & purification; D006859:Hydrogen / Q000737:chemistry*; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation; D008958:Models, Molecular; D011487:Protein Conformation; D017510:Protein Folding
chemical_list: D006859:Hydrogen; D045304:Cytochromes c