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

Abstract

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 N^H 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.