NMR-based amide hydrogen-deuterium exchange measurements for complex membrane proteins: Development and critical evaluation

A method for measuring site-specific amide hydrogen-deuterium exchange rate s for membrane proteins in bilayers is reported and evaluated. This method represents an adaptation and extension of the approach of Dempsey and co-wo rkers (Biophys, J. 70, 1777-1788 (1996)) and is based on reconstituting N-1 5-labeled membrane proteins into phospholipid bilayers, followed by lyophil ization and rehydration with D2O or H2O (control), Following incubation for a time t under hydrated conditions, samples are again lyophilized and then solubilized in an organic solvent system, where H-1-N-15 HSQC spectra are recorded. Comparison of spectra from D2O-exposed samples to spectra from co ntrol samples yields the extent of the H-D exchange which occurred in the b ilayers during time t. Measurements are site specific if specific N-15 labe ling is used, The first part of this paper deals with the search for a suit able solvent system in which to solubilize complex membrane proteins in an amide "exchange-trapped" form for NMR quantitation of amide peak intensitie s. The second portion of the paper documents application of the overall pro cedure to measuring site-specific amide exchange rates in diacylglycerol ki nase, a representative integral membrane protein. Both the potential useful ness and the significant limitations of the new method are documented. (C) 2000 Academic Press.