title: NMR-Based amide hydrogen-deuterium exchange measurements for complex membrane proteins: development and critical evaluation.
abstract: A method for measuring site-specific amide hydrogen-deuterium exchange rates for membrane proteins in bilayers is reported and evaluated. This method represents an adaptation and extension of the approach of Dempsey and co-workers (Biophys. J. 70, 1777-1788 (1996)) and is based on reconstituting (15)N-labeled membrane proteins into phospholipid bilayers, followed by lyophilization and rehydration with D(2)O or H(2)O (control). Following incubation for a time t under hydrated conditions, samples are again lyophilized and then solubilized in an organic solvent system, where (1)H-(15)N HSQC spectra are recorded. Comparison of spectra from D(2)O-exposed samples to spectra from control samples yields the extent of the H-D exchange which occurred in the bilayers during time t. Measurements are site specific if specific (15)N labeling is used. The first part of this paper deals with the search for a suitable solvent system in which to solubilize complex membrane proteins in an amide "exchange-trapped" form for NMR quantitation of amide peak intensities. The second portion of the paper documents application of the overall procedure to measuring site-specific amide exchange rates in diacylglycerol kinase, a representative integral membrane protein. Both the potential usefulness and the significant limitations of the new method are documented.
authors.affiliation: Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106-4970, USA.
authors.forename: L
authors.initials: L
authors.lastname: Czerski
authors.forename: O
authors.initials: O
authors.lastname: Vinogradova
authors.forename: C R
authors.initials: CR
authors.lastname: Sanders
mesh_terms: D000577:Amides; D003903:Deuterium; D019852:Diacylglycerol Kinase / Q000737:chemistry*; D004926:Escherichia coli; D006801:Humans; D006859:Hydrogen; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008565:Membrane Proteins / Q000737:chemistry*
chemical_list: D000577:Amides; D008565:Membrane Proteins; D006859:Hydrogen; D003903:Deuterium; D019852:Diacylglycerol Kinase