title: Pulsed hydrogen exchange and electrospray charge-state distribution as complementary probes of protein structure in kinetic experiments: implications for ubiquitin folding.
abstract: The possible involvement of "hidden" kinetic intermediates in the apparent two-state folding of some proteins is currently a matter of debate. This study uses time-resolved electrospray ionization (ESI) mass spectrometry with on-line pulsed hydrogen-deuterium exchange (HDX) for monitoring the refolding of acid/methanol-denatured ubiquitin. It is demonstrated that the ESI charge-state distribution (CSD) and the extent of HDX represent nonredundant probes of the protein structure in solution. When considered in isolation, the data provided by both of these probes are consistent with a two-state behavior, involving only denatured ubiquitin D and refolded protein F. However, a careful comparison of the CSD and HDX kinetics reveals the presence of an additional species, exhibiting a CSD like the folded protein but showing non-native HDX characteristics. This kinetic intermediate, D*, is in rapid equilibrium with D, such that the overall reaction is consistent with the mechanism D <--> D* --> F. The results of this work suggest that the occurrence of transient intermediates may be more widespread than commonly thought, especially in cases where a cursory analysis indicates two-state behavior.
authors.affiliation: Department of Chemistry, The University of Western Ontario, London, Ontario, N6A 5B7, Canada.
authors.forename: Jingxi
authors.initials: J
authors.lastname: Pan
authors.forename: Derek J
authors.initials: DJ
authors.lastname: Wilson
authors.forename: Lars
authors.initials: L
authors.lastname: Konermann
mesh_terms: D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D003903:Deuterium; D006859:Hydrogen; D007700:Kinetics; D011487:Protein Conformation; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D025801:Ubiquitin / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D011506:Proteins; D025801:Ubiquitin; D006859:Hydrogen; D003903:Deuterium