NMR-Based Amide Hydrogen–Deuterium Exchange Measurements for Complex Membrane Proteins: Development and Critical Evaluation
✍ Scribed by Lech Czerski; Olga Vinogradova; Charles R Sanders
- Book ID
- 102600285
- Publisher
- Elsevier Science
- Year
- 2000
- Tongue
- English
- Weight
- 272 KB
- Volume
- 142
- Category
- Article
- ISSN
- 1090-7807
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✦ Synopsis
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.