Direct assignment of 13C solid-state NMR signals of TFoF1 ATP synthase subunit c-ring in lipid membranes and its implication for the ring structure

F o F 1 -ATP synthase catalyzes ATP hydrolysis/synthesis coupled with a transmembrane H + translocation in membranes. The F o c -subunit ring plays a major role in this reaction. We have developed an assignment strategy for solid-state 13 C NMR (ssNMR) signals of the F o c -subunit ring of thermophilic Bacillus PS3 (TF o c -ring, 72 residues), carrying one of the basic folds of membrane proteins. In a ssNMR spectrum of uniformly 13 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 α(i+1) –C′C α(i) correlation and dipolar assisted rotational resonance (DARR) experiments were performed under magic-angle sample spinning. On the basis of inter- and intra-residue 13 C– 13 C chemical shift correlations, 97% of C α , 97% of C β 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 o 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 o 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.