Determination of Local Protein Structure by Spin Label Difference 2D NMR: The Region Neighboring Asp61 of Subunit c of the F1F0 ATP Synthase

Mark E. Girvin, Robert H. Fillingame

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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 hairpinlike folding is predicted for the native protein in the multisubunit transmembrane F0 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 within 15 Å 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 hydrogenbonding 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°) 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.

Original languageEnglish (US)
Pages (from-to)1635-1645
Number of pages11
JournalBiochemistry
Volume34
Issue number5
DOIs
StatePublished - Feb 1995

ASJC Scopus subject areas

  • Biochemistry

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