After purification, protein samples were reduced using dithiothreitol, and then labeled using a thiol-specific fluorescent probe, fluorescein-5-maleimide. Under this experimental condition, only proteins that contain disulfide bond in their native structure would be labeled and fluoresce. In each experiment, we have also included internal positive and negative controls. In the positive control, proteins were purified in the absence of IAM and then reduced with DTT before labeling. Therefore, all proteins that contained Cys, either in the free thiol or disulfide bond form, would be labeled. In the negative control, IAM was present during protein purification, but the purified samples were not reduced before labeling. Therefore, no protein should be labeled. We used 4 Cys-pair reporters established previously to Gefitinib evaluate the tertiary structure of AcrBP223G. In addition, we have created a new reporter Cys pair in this study, A216C–I234C. This pair exists in the loop. We used it to detect potential conformational changes in the loop. We have confirmed that disulfide bond formed between C216 and C234 when they were introduced into a Cys-less AcrB construct, in which the two intrinsic Cys in the sequence of AcrB were replaced with Ala. In addition, the mutations and formation of disulfide bond had no effect on the drug efflux activity of AcrB. Two pictures of SDS-PAGE gels were shown for each protein, one visualized under fluorescence light without staining and the other stained with coomassie blue dye. Coomassie blue stain revealed the loading amount in each lane, while the fluorescence image reflected the formation of disulfide bond. As discussed above, the first two lanes were positive and negative controls, respectively. The third lane revealed the presence of disulfide bond. For each reporter Cys pair tested in the presence of P223G mutation, the fluorescence in the third lane was comparable to that of the WT AcrB, indicating that the disulfide formed similarly as in the WT protein. Furthermore, the new reporter Cys pair, 216–234, showed that the structure of the loop did not change significantly compared to that of the WT protein. These results suggested that the P223G mutation did not change the tertiary structure of the protein. In an effort to understand the AcrB trimerization process and the inter-subunit interactions that lead to trimerization, we have previously developed a disulfide trapping method to characterize AcrB tertiary structure in membrane and created a well folded monomeric AcrB mutant. Based on the crystal structure of AcrB, each subunit contains a protruding loop, which extends deep into a tunnel in the neighboring subunit. The loopto-tunnel interaction is apparently very important to the formation and stability of AcrB trimer. The unique backbone structure of P223 induces the loop to form a kink close to the tip, which suggests that it may fun ction as a wedge to lock the loop in place and stabilize the trimer structure.