H⁺-ATP synthase subunit c

H⁺-ATP synthase is an important enzyme in the energy transduction system in organisms. Subunit c is a hydrophobic protein composed of 79 amino-acids, and plays a key role by transporting protons across the membrane. Our current goal is to assign the signals and analyze the secondary structure based on the assigned chemical shifts.

One of the characteristics of transmembrane proteins is the high occurence of hyddrophobic amino acids such as leucine and valine. Because signals of solid-state NMR are broad and because often times the transmamebrane region all takes alpha-helical conformation, it is very difficult to resolve those signals. Conventionally, amino-acid or site-specific isotope labeling have been used to avoid the difficulty, but it requires a series of samples labeled differently for complete signal assignment. Also, the number of samples to be prepared would increase with increasing molecular weight of the protein. This is time/cost ineffective.

We are trying to assign signals and elucidate the secondary structure of the protein by using only one uniformly ¹³C and ¹⁵N-labeled sample. Another challenge is to determine arrangement of the transmembrane region in the lipid bilayer and precise topology from inter-nuclear distance information obtained from this uniformly-labeled sample.


Using two- and three-dimensional magic-angle spinning spectroscopy, we assigned many ¹³C and ¹⁵N signals: 78% of C-alpha, 72% of C-beta, 62% of Co and 61% of amide nitrogens. In addition, ¹³C-¹³C correlation spectra were fitted by simulated spectra generated based on predicted chemical shifts and peak intensities from structure models. This revealed that the subunit c in the solid-state has two helices with a loop structure in between helices and at c-terminus. Currently, we are studying structure of subunit c that is reconstituted into the lipid bilayer condition using similar approach.