The ultimate goal of this proposal is to set the stage for significant advances in structural characterization of membrane proteins by solution NMR. To approach membrane proteins, we turn to a novel method in solution NMR of encapsulated proteins dissolved in low viscosity fluids. Using the largely helical KcsA potassium channel and the largely beta-sheet OmpA porin as model systems, we will refine the methods of reverse micelle encapsulation of membrane proteins. Then, we will take advantage of a remarkable library of more than 500 integral membrane proteins, which is available to us through collaboration with Dr. von Heijne and coworkers. Over one hundred proteins express at sufficient levels for high throughput production and biophysical characterization. We will select about two dozen of small, high-expressing membrane proteins that will be incorporated into reverse micelles and tested for structural integrity. Three dimensional structures of the best behaved protein(s) will be determined, validating the feasibility of applying triple resonance NMR methods in reverse micelles to membrane proteins. Gathered statistics of successful encapsulation will help guide future high-throughput structural studies of membrane proteins. ? ?
