Direct evidence for a two-state protein unfolding transition from hydrogen-deuterium exchange, mass spectrometry, and NMR

TitleDirect evidence for a two-state protein unfolding transition from hydrogen-deuterium exchange, mass spectrometry, and NMR
Publication TypeJournal Article
Year of Publication1996
AuthorsYi, Q., & Baker D.
JournalProtein science
Volume5
Issue6
Pagination1060-6
Date Published1996 Jun
ISSN0961-8368
KeywordsBacterial Proteins, Binding Sites, Deuterium, Hydrogen-Ion Concentration, Immunoglobulin G, Magnetic Resonance Spectroscopy, Mass Spectrometry, Models, Chemical, Peptostreptococcus, Primary Publication, Protein Conformation, Protein Folding, Protein Structure, Tertiary, Protons, Temperature, Time Factors
Abstract

We use mass spectrometry in conjunction with hydrogen-deuterium exchange and NMR to characterize the conformational dynamics of the 62-residue IgG binding domain of protein L under conditions in which the native state is marginally stable. Mass spectra of protein L after short incubations in D2O reveal the presence of two distinct populations containing different numbers of protected protons. NMR experiments indicate that protons in the hydrophobic core are protected in one population, whereas all protons are exchanged for deuterons in the other. As the exchange period is increased, molecules are transferred from the former population to the latter. The absence of molecules with a subset of the core protons protected suggests that exchange occurs in part via a highly concerted transition to an excited state in which all protons exchange rapidly with deuterons. A steady increase in the molecular weight of the population with protected protons, and variation in the exchange rates of the individual protected protons indicates the presence of an additional exchange mechanism. A simple model in which exchange results from rapid (> 10(5)/s) local fluctuations around the native state superimposed upon transitions to an unfolded excited state at approximately 0.06/s is supported by qualitative agreement between the observed mass spectra and the mass spectra simulated according to the model using NMR-derived estimates of the proton exchange rates.

Alternate JournalProtein Sci.
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