Helical structure and folding of subunit c of F1F0 ATP synthase: 1H NMR resonance assignments and NOE analysis.
Subunit c of the H(+)-transporting F1F0 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 F0. We report here the initial results of a structural study of purified subunit c in a chloroform-methanol-water (4:4:1) solvent mixture using standard two-dimensional NMR techniques. The spin systems of 78 of the 79 amino acid side chains have been assigned to residue type, and 44 of these have been assigned to specific residues in the sequence. Stretches of alpha-helical secondary structure were observed for Asp7-ILe26 in the first proposed transmembrane helix, and for Arg50-Ile55 and Ala67-Val78 in the second proposed transmembrane helix. Nuclear Overhauser effects (NOEs) were observed between residues at both ends of the predicted transmembrane helices. The intensities of the NOEs between helix-1 and helix-2 were not diminished by mixing of 2H-subunit c with 1H-subunit c, and therefore the NOEs must be due to intramolecular, rather than intermolecular, interactions. Hence the purified protein must fold as a hairpin in this solvent system, just as it is thought to fold in the lipid bilayer of the membrane. In native F0, dicyclohexylcarbodiimide reacts specifically with Asp61 in the second transmembrane helix of subunit c, and the rate of this reaction is reduced by substitution of Ile28 by Thr on the first transmembrane helix. The I28T substitution is shown here to alter the chemical shifts of protons at and around Asp61. This observation provides a further indication that subunit c may fold in chloroform-methanol-water solvent much like it does in the membrane.
Girvin
M E
ME
Department of Biomolecular Chemistry, University of Wisconsin, Madison 53706.
Fillingame
R H
RH
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D004024:Dicyclohexylcarbodiimide; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D017510:Protein Folding; D017433:Protein Structure, Secondary; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000302:isolation & purification; D012997:Solvents
D000596:Amino Acids; D012997:Solvents; D004024:Dicyclohexylcarbodiimide; D006180:Proton-Translocating ATPases