FIGURE 2.

Disulfide cross-link formation in F₁F₀ ATP synthase complexes containing engineered E. coli peripheral stalks. A, membranes were prepared in the presence of 5 mm Tris (2-carboxyethyl) phosphine from strains KM2/pSBC127 (b_V5), KM2/pSBC128 (b_I76C,V5), KM2/pSBC129 (b_R83C,V5), KM2/pSBC130 (b_A90C,V5), and KM2/pSBC131 (b_E97C,V5). Cross-link formation was induced by diluting samples to 5 mg/ml and adding Cu²⁺ to a final concentration of 500 μm. The reactions were carried out at 37 °C with shaking for 2 min, and the cross-linking reaction was quenched by adding NEM. The fraction dimer was calculated for each sample by dividing the dimer band by the sum of the monomer and dimer bands. The vertical lines indicate the removal of intervening lanes from the gel in silico. The samples with a low probability of being identical are indicated (*, p < 0.05; **, p < 0.01; ***, p < 0.001; n = 6). B, samples were prepared and cross-linked essentially as described for A with the exception that the Cu²⁺ concentration was reduced to 50 μm, and the reaction time was increased to 10 min.