1ATY

DETERMINATION OF LOCAL PROTEIN STRUCTURE BY SPIN LABEL DIFFERENCE 2D NMR: THE REGION NEIGHBORING ASP61 OF SUBUNIT C OF THE F1FO ATP SYNTHASE

1ATY の概要

• エントリーDOI: 10.2210/pdb1aty/pdb
• 分子名称: F1F0 ATP SYNTHASE (SUBUNIT C) (1 entity in total)
• 機能のキーワード: membrane protein
• 由来する生物種: Escherichia coli
• 細胞内の位置: Cell inner membrane; Multi-pass membrane protein: P68699
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 8291.13

構造登録者

Girvin, M.E.,Fillingame, R.H. (登録日: 1994-10-05, 公開日: 1994-12-20, 最終更新日: 2024-05-22)

主引用文献

Girvin, M.E.,Fillingame, R.H.
Determination of local protein structure by spin label difference 2D NMR: the region neighboring Asp61 of subunit c of the F1F0 ATP synthase.
Biochemistry, 34:1635-1645, 1995

PubMed Abstract: Purified subunit c from the H(+)-transporting F1F0 ATP synthase of Escherichia coli folds as an antiparallel pair of extended helices in a solution of chloroform-methanol-water. A similar hairpin-like folding is predicted for the native protein in the multisubunit transmembrane Fo sector of the ATP synthase. A single Cys variant (A67C) of subunit c was created and modified with a maleimido-PROXYL [[3-(maleimidomethyl)-2,2,5,5-tetramethyl-1-pyrrolidinyl]oxy] spin label. Pairs of 1H 2D correlation and NOE spectra were collected with the nitroxide oxidized (paramagnetic) and reduced (diamagnetic). The pairs of spectra were subtracted, yielding difference spectra containing only cross-peaks from 1H within 15 A of the spin label. These greatly simplified spectra were easily analyzed to provide complete assignments for residues 10-25 and 52-79 of the protein, 150 NOE distance restraints, and 27 hydrogen-bonding restraints. The chemical shifts and NOE patterns observed in the derivatized mutant were virtually identical to those which were resolved in the unmodified wild-type protein, strongly suggesting that the spin label was not perturbing the protein structure. The restaints enabled us to calculate a detailed structure for this region of subunit c. The structure consisted of two gently curved helices, crossing at a slight (30 degrees) angle. The C-terminal helix was disrupted from Val60 to Ala62 near the essential Pro64. Asp61, the residue thought to undergo protonation--deprotonation with each H+ transported across the membrane, was in ver der Waals contact with Ala24. The proximity of these residues had been predicted from mutant analyses, where H+ translocation was retained on moving the Asp from position 61 to 24.

PubMed: 7849023
DOI: 10.1021/bi00005a020

実験手法

SOLUTION NMR