Direct assignment of 13 C solid-state NMR signals of TF o F 1 ATP synthase subunit c -ring in lipid membranes and its implication for the ring structure
 

Direct assignment of 13 C solid-state NMR signals of TF o F 1 ATP synthase subunit c -ring in lipid membranes and its implication for the ring structure

Abstract

FoF1-ATP synthase catalyzes ATP hydrolysis/synthesis coupled with a transmembrane H+ translocation in membranes. The Fo c-subunit ring plays a major role in this reaction. We have developed an assignment strategy for solid-state 13C NMR (ssNMR) signals of the Fo c-subunit ring of thermophilic Bacillus PS3 (TFo c-ring, 72 residues), carrying one of the basic folds of membrane proteins. In a ssNMR spectrum of uniformly 13C-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 Cα(i+1)â??Câ?²Cα(i) correlation and dipolar assisted rotational resonance (DARR) experiments were performed under magic-angle sample spinning. On the basis of inter- and intra-residue 13Câ??13C chemical shift correlations, 97% of Cα, 97% of Cβ and 92% of Câ?² 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 TFo c-ring structure were examined. The DARR spectra at 15Â*ms mixing time are essentially similar to each other in saturated and unsaturated lipid membranes, suggesting that TFo 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.



Source: Journal of Biomolecular NMR