DETERMINATION OF LOCAL PROTEIN-STRUCTURE BY SPIN-LABEL DIFFERENCE 2D NMR - THE REGION NEIGHBORING ASP61 OF SUBUNIT-C OF THE F1F0 ATP SYNTHASE

Purified subunit c from the H+-transporting F1F0 ATP synthase of Esche richia coli folds as an antiparallel pair of extended helices in a sol ution of chloroform-methanol-water. A similar hairpin-like folding is predicted for the native protein in the multisubunit transmembrane F-0 sector of the ATP synthase. A single Cys variant (A67C) of subunit c was created and modified with a maleimido-PROXYL idomethyl)-2,2,5,5-te tramethyl-1-pyrrolidinyl]oxy] spin label. Pairs of H-1 2D correlation and NOE spectra were collected with the nitroxide oxidized (paramagnet ic) and reduced (diamagnetic). The pairs of spectra were subtracted, y ielding difference spectra containing only cross-peaks from H-1 within 15 Angstrom 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-bo nding restraints. The chemical shifts and NOE patterns observed in the derivatized mutant were virtually identical to those which were resol ved in the unmodified wild-type protein, strongly suggesting that the spin label was not perturbing the protein structure. The restaints ena bled 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 Val 60 to Ala62 near the essential Pro64. Asp61, the residue thought to un dergo protonation-deprotonation with each H+ transported across the me mbrane, was in ver der Waals contact with Ala24. The proximity of thes e residues had been predicted from mutant analyses, where H+ transloca tion was retained on moving the Asp from position 61 to 24.