| In this work the feasibility of sequential, site-specific assignments of uniformly 13C, 15N enriched helical integral membrane proteins using solid state Nuclear Magnetic Resonance (ssNMR) spectroscopy was established. Recent technical advances in ssNMR make it a promising method for exploring membrane proteins and other non-soluble or large biological systems. The prerequisite of NMR based structure determination for any protein is the assignment of resonance peaks; to date, only a few complete uniformly or extensively 13C, 15N enriched protein assignments have been published.; Two membrane proteins were investigated: the c subunit of E. coli ATP synthase and S. lividans KcsA potassium channel. For the c subunit of ATP synthase, the effect of sample preparation on structure was studied, where ssNMR served as a superb tool to observe secondary structure changes. Based on literature, the c subunit is an alpha-helical protein, however some unexpected beta-sheet structure is observed in several protein preparations including (1) detergent micelles precipitated with MPD, (2) bicelles precipitated with polyethylene glycol (PEG), (3) lipid vesicles pelleted by ultracentrifugation, and (4) nanodiscs precipitated with PEG. The amount of beta-sheet protein appears to depend on the purity of the protein, an issue which requires further investigation.; The KcsA potassium channel was precipitated in n-Dodecyl-D-maltoside detergent micelles with PEG 1,000 near neutral pH. Partial site-specific sequential 13C and 15N assignments were determined, including both backbone and some side chains atoms near and at the selectivity filter region of the protein: V76-D80 and P83-C90. Since the secondary structure is not all helical in this region of the protein, the chemical shifts of some residues are far shifted from the typical helical residues, and provide a good fingerprint for the sample. Residue types were confirmed by side chain assignments. The experimental strategy included a combination of 2D and 3D ssNMR techniques at 600 and 750 MHz. The availability of these specific assignments open the door for membrane protein structure determination and biophysical characterization by ssNMR. |
