HELICAL STRUCTURE AND FOLDING OF SUBUNIT-C OF F(1)F(0) ATP SYNTHASE -H-1-NMR RESONANCE ASSIGNMENTS AND NOE ANALYSIS

Subunit c of the H+-transporting F1F(o) ATP synthase (EC 3.6.1.34) is thought to fold across the membrane as a hairpin of two alpha-helices and function as a key component of the H+-translocase of F(o). We repo rt here the initial results of a structural study of purified subunit c in a chloroform-methanol-water (4:4:1) solvent mixture using standar d two-dimensional NMR techniques. The spin systems of 78 of the 79 ami no acid side chains have been assigned to residue type, and 44 of thes e have been assigned to specific residues in the sequence. Stretches o f alpha-helical secondary structure were observed for Asp7-Ile26 in th e first proposed transmembrane helix, and for Arg50-Ile55 and Ala67-Va l78 in the second proposed transmembrane helix. Nuclear Overhauser eff ects (NOEs) were observed between residues at both ends of the predict ed transmembrane helices. The intensities of the NOEs between helix-1 and helix-2 were not diminished by mixing of H-2-subunit c with H-1-su bunit c, and therefore the NOEs must be due to intramolecular, rather than intermolecular, interactions. Hence the purified protein must fol d as a hairpin in this solvent system, just as it is thought to fold i n the lipid bilayer of the membrane. In native F(o), dicyclohexylcarbo diimide reacts specifically with Asp61 in the second transmembrane hel ix of subunit c, and the rate of this reaction is reduced by substitut ion of Ile28 by Thr on the first transmembrane helix. The I28T substit ution is shown here to alter the chemical shifts of protons at and aro und Asp61. This observation provides a further indication that subunit c may fold in chloroform-methanol-water solvent much like it does in the membrane.