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 15N-labeled membrane proteins into phospholipid bilayers, followed by lyophilization 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 1H–15N HSQC spectra are recorded. Comparison of spectra from D2O-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 15N 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.