HAIRPIN FOLDING OF SUBUNIT-C OF F(1)F(0) ATP SYNTHASE - H-1 DISTANCE MEASUREMENTS TO NITROXIDE-DERIVATIZED ASPARTYL-61

Subunit c from the F1F(o) ATP synthase of Escherichia coli folds in a hairpinlike structure of two a-helices in a solution of chloroform-met hanol-H2O, and thus resembles the structure predicted for the folded p rotein in the membrane. The relevance of the structure in solution to the native structure was demonstrated. Asp61 in the second helical arm was shown to retain its unique reactivity with dicyclohexylcarbodiimi de (DCCD) in chloroform-methanol-H2O solution. Further, the protein pu rified from the Ile28-->Thr DCCD-resistant mutant proved to be less re active with DCCD in solution. This suggested that the protein folded w ith Ile28 of the first helical arm close to Asp61 in the second helica l arm. Subunit c in wild-type E. coli membranes was specifically label ed with a nitroxide analog of DCCD (NCCD), and the derivative protein was purified. DQF COSY spectra were recorded, and the distances betwee n the paramagnetic nitroxide and resolved protons in the spectra were calculated based upon paramagnetic broadening of the H-1 resonances. T he paramagnetic contribution to T2 relaxation in the NCCD-labeled samp le was calculated by an iterative computer-fitting method, where a con trol spectrum of a phenylhydrazine-reduced sample was broadened until the line shape of one-dimensional slices through each COSY cross-peak maximally mimicked the line shape of the paramagnetic sample. The dist ances calculated from paramagnetic broadening indicate that Ala24 and Ala25 in helix-1 lie close (ca. 12 angstrom) to the derivatized Asp61 in helix-2. A model for the interaction of helices in the NCCD-modifie d protein was generated by restrained molecular mechanics and molecula r dynamics using 25 distances of <10-20 angstrom derived from paramagn etic broadening in combination with 15 long-range nuclear Overhauser e nhancement (NOE) restraints (2-5 angstrom) for distances between helic es and the 89 intrahelical NOEs that defined helical structure in the DCCD-modified protein.