The overall goals of this project are to develop and apply new methodology to obtain the atomic-resolution structure of membrane proteins by solid-state NMR (SSNMR). The new techniques include sample preparation, isotopic labeling strategies, high-field (750 MHz 1H frequency) magic-angle spinning NMR spectroscopy and multidimensional dipolar recoupling pulse sequences. Our preliminary data demonstrate that we can obtain high quality 2D and 3D SSNMR spectra of E. coli cytochrome bo3 ubiquinol oxidase, a large (~144 kDa) integral membrane protein complex. The enzyme was uniformly labeled with 13C and 15N, then purified and pelleted with endogenous membrane lipids. Excellent spectra can be obtained with approximately 50 nmol (approximately 8 mg) of purified protein (using 12 to 36 hours of measurement time for 2D experiments, or 24 to 96 hours for 3D experiments). The protein-lipid complex maintains the protein in an active, native conformation and provides a stabilizing environment for the protein. The sample shows no evidence of degradation after weeks of NMR data acquisition. Our data demonstrate that SSNMR has the immediate potential to interrogate selected parts of very large uniformly 13C,15N -labeled membrane proteins, and to provide complete 3D structures of smaller membrane proteins (approximatley 200 residues). To realize this potential is the goal of the proposed project. Improved methods for structure determination by SSNMR may relieve a serious bottleneck in the membrane protein structure determination pipeline.
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