Signal assignment and secondary structure analysis of a uniformly [13C, 15N]-labeled membrane protein, H+-ATP synthase subunit c, by magic-angle spinning solid-state NMR

Signal assignment and secondary structural analysis of uniformly [13C, 15N] labeled H+-ATP synthase subunit c from E. coli (79 residues) in the solid state were carried out by two- and three-dimensional solid-state NMR under magic-angle spinning. The protein took on a unique structure even in the solid state from the 13C linewidths of about 1.7 ppm. On the basis of several inter- and intra-residue 13C–13C and 13C–15N chemical shift correlations, 78% of $${\rm C}^{\upalpha}$$ , 72% of $${\rm C}^{\upbeta}$$ , 62% of C′ and 61% of NH signals were assigned, which provided the secondary structure information for 84% of the 79 residues. Here, inter-residue correlations involving Gly, Ala, Pro and side-chains and a higher resolution in the 3D spectrum were significantly useful for the sequence specific assignment. On top of this, the 13C–13C correlation spectra of subunit c was analyzed by reproducing experimental cross peaks quantitatively with chemical shift prediction and signal-intensity calculation based on the structure. It revealed that the subunit c in the solid state could be specified by $$\upalpha$$ -helices with a loop structure in the middle (at sequence 41–45) as in the case of the solution structure in spite of additional extended conformations at 76–79 at the C-terminus.