FIGURE 8.
Probing the conformational changes at the central channel of the Rho mutants. Locations of different single cysteine derivatives (left panels) and corresponding modified Stern-Volmer plots obtained from the acrylamide quenching of the fluorescein molecule attached to each of the cysteine derivatives are shown in A–E. Due to non-linear nature of the plots, we used the modified Stern-Volmer equations to fit the curves as described under ”Materials and Methods.“ We calculated the KSV from the initial slopes of the curves, which approximately equals to the respective dynamic quenching constants. The P-, Q-, R-loops are indicated in the structure with the single cysteines as spheres. F, 12% (w/v) SDS-PAGE showing the formation of inter-molecular disulfide bonds between the cysteines of neighboring molecules using copper phenanthroline (Cu-P) as a reducing agent.