Subunit c from the F1Fo ATP synthase of Escherichia coli folds in a hairpinlike structure of two α-helices in a solution of chloroform-methanol-H2O, and thus resembles the structure predicted for the folded protein 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 dicyclohexylcarbodiimide (DCCD) in chloroform-methanol-H2O solution. Further, the protein purified from the Ile28→Thr DCCD-resistant mutant proved to be less reactive with DCCD in solution. This suggested that the protein folded with Ile28 of the first helical arm close to Asp61 in the second helical arm. Subunit c in wild-type E. coli membranes was specifically labeled with a nitroxide analog of DCCD (NCCD), and the derivative protein was purified. DQF COSY spectra were recorded, and the distances between the paramagnetic nitroxide and resolved protons in the spectra were calculated based upon paramagnetic broadening of the 1H resonances. The paramagnetic contribution to T2 relaxation in the NCCD-labeled sample was calculated by an iterative computer-fitting method, where a control 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 distances calculated from paramagnetic broadening indicate that Ala24 and Ala25 in helix-1 lie close (ca. 12 Å) to the derivatized Asp61 in helix-2. A model for the interaction of helices in theNCCD-modified protein was generated by restrained molecular mechanics and molecular dynamics using 25 distances of <10–20 Å derived from paramagnetic broadening in combination with 15 long-range nuclear Overhauser enhancement (NOE) restraints (2–5 Å) for distances between helices and the 89 intrahelical NOEs that defined helical structure in the DCCD-modified protein.
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