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Cytochrome c folding kinetics studied by time-resolved electrospray ionization mass spectrometry

TitleCytochrome c folding kinetics studied by time-resolved electrospray ionization mass spectrometry
Publication TypeJournal Article
Year of Publication1997
AuthorsKonermann, L, Collings, BA, Douglas, DJ
JournalBiochemistry
Volume36
Pagination5554-5559
Date PublishedMay
Type of ArticleArticle
ISBN Number0006-2960
KeywordsCOMPLEX, denaturation, FERRICYTOCHROME-C, IRON BLEOMYCIN, LIGANDS, MECHANISM, PATHWAYS, PROBING CONFORMATIONAL-CHANGES, PROTEINS, SPECTRA
Abstract

A new method for studying the folding kinetics of proteins is described. The method combines a continuous flow mixing technique with an electrospray mass spectrometer. Different protein conformations in solution are detected by the different charge states they produce during electrospray ionization. Unfolded proteins generally have more accessible protonation sites and give higher charge states than native proteins. The method is applied to study the refolding of acid-denatured cytochrome c. Global data analysis is used to obtain the exponential lifetimes which are associated with the refolding process. The kinetics can be described by two lifetimes of 0.17 +/- 0.02 and 8.1 +/- 0.9 s which are in accordance with the results of stopped flow experiments previously described in the literature. These lifetimes are associated with roughly 90 and 10% of the total intensity changes in the mass spectrum, respectively, and most likely reflect fast and slow refolding subpopulations of cytochrome c in solution.

URL<Go to ISI>://A1997WY06300029