Direct assignment of C solid-state NMR signals of TFF ATP synthase subunit c-ring in lipid membranes and its implication for the ring structure.

FF-ATP synthase catalyzes ATP hydrolysis/synthesis coupled with a transmembrane H translocation in membranes. The F c-subunit ring plays a major role in this reaction. We have developed an assignment strategy for solid-state C NMR (ssNMR) signals of the F c-subunit ring of thermophilic Bacillus PS3 (TF c-ring, 72 residues), carrying one of the basic folds of membrane proteins. In a ssNMR spectrum of uniformly C-labeled sample, the signal overlap has been a major bottleneck because most amino acid residues are hydrophobic. To overcome signal overlapping, we developed a method designated as COmplementary Sequential assignment with MInimum Labeling Ensemble (COSMILE). According to this method, we generated three kinds of reverse-labeled samples to suppress signal overlapping. To assign the carbon signals sequentially, two-dimensional C-C'C correlation and dipolar assisted rotational resonance (DARR) experiments were performed under magic-angle sample spinning. On the basis of inter- and intra-residue C-C chemical shift correlations, 97% of C, 97% of C and 92% of C' signals were assigned directly from the spectra. Secondary structure analysis predicted a hairpin fold of two helices with a central loop. The effects of saturated and unsaturated phosphatidylcholines on TF c-ring structure were examined. The DARR spectra at 15 ms mixing time are essentially similar to each other in saturated and unsaturated lipid membranes, suggesting that TF c-rings have similar structures under the different environments. The spectrum of the sample in saturated lipid membranes showed better resolution and structural stability in the gel state. The C-terminal helix was suggested to locate in the outer layer of the c-ring.