FIGURE 7.

Cross-linking Atto590 labeling on E208C and A281C in dimeric MsbA-cl. A, when ISOVs containing A281C MsbA-cl were loaded on an SDS-polyacrylamide gel without the reducing agent DTT in the SDS sample-loading buffer and analyzed by Western blot, high molecular weight dimers were observed due to spontaneous cysteine cross-linking. These dimers were absent for MsbA-cl (Cys-less). Cysteine cross-linking was enhanced by incubation with the oxidizing agent copper phenanthroline (CuPhen), although cross-linking disappeared on the inclusion of DTT in the SDS sample-loading buffer. B, under no nucleotide conditions, E208C MsbA-cl (10) or A281C MsbA-cl in ISOVs was subjected to inter-molecular cysteine cross-linking, and MsbA-specific bands (monomers (M)/dimers (D)) were detected on Western blot probed with an anti-penta-His antibody (10). The approximate molecular mass marker positions are shown alongside. C, high saturating levels of Cys-Cys cross-linking were observed for A281C MsbA-cl in the no nucleotide condition. As the reduction in this high intensity after the transition from inward-facing to outward-facing MsbA is associated with a poor signal-to-noise ratio, we combined the reduction in A281C/A281C′ cross-linking with measurements on the increased accessibility of free A281C to the thiol-reactive fluorescent probe Atto590. To validate this method, we re-assayed A281C MsbA-cl and E208C MsbA-cl in the no nucleotide condition. On Coomassie Brilliant Blue staining of the gel, high molecular weight bands corresponding to E208C/E208C′ and A281C/A281C′ Cys-Cys cross-linked dimers (D) were observed, whereas such dimers were not obtained for MsbA-cl. A small but reproducible difference in migration was observed between dimers for the two mutants, which is attributed to anomalous SDS binding. The intensity of dimers for A281C MsbA-cl was found to be higher than for E208C MsbA-cl, consistent with the notion that the MsbA dimer will be in the inward-facing state in the absence of added nucleotides. D, when the same gel was visualized under UV light (prior to Coomassie-staining) only ISOVs containing E208C MsbA-cl or A281C MsbA-cl had the monomers (M) labeled with Atto590; labeled monomers were not observed for control and MsbA-cl ISOVs. Consistent with the enhanced dimer intensity for A281C MsbA-cl, the monomer Atto590-labeling intensity was reduced for this mutant compared with E208C MsbA-cl. The images in C and D pertain to one and the same gel/experiment, repeated at least three times, and were processed in parallel and cropped to increase clarity of presentation.