AcrAB-TolC and its homologues, members of the resistancenodulation-cell division transporter family, are major efflux systems that make Gram-negative bacteria resistant against a wide range of cytotoxic compounds. The structure of AcrB has been solved by x-ray crystallography in both the apo and substrate-bound conformations. Based on the crystal structure of AcrB, a conformational cycling model for drug transport has been proposed. However, crystal structures can not provide insight into the biogenesis process of an AcrB trimer. Recently, we have created a monomeric AcrB mutant, AcrBDloop, in which we deleted 17 residues from a protruding loop . The loop is obviously important for inter-subunit interactions, as it penetrates deep into a tunnel in the neighboring subunit. While at the same time, it stretches away from the rest of the polypeptide chain, not making tertiary contact with any residues from the same subunit. We found that AcrBDloop completely lost its transport activity and failed to assembly into a trimer, while had a similar tertiary structure as subunits in the AcrB trimer. These results indicated that monomeric AcrB was capable of folding independently, suggesting that oligomerization of AcrB occurred through a three-stage pathway, in which nascent polypeptide chains first folded independently into monomers, which then assembled into functional trimers. To further probe the role and structural flexibility of the protruding loop during AcrB trimerization, we mutated a conserved Pro and characterized the structure and function of the resultant mutants. We found that replacing P223 with other residues drastically decreased the stability of the AcrB trimer and caused a loss of function, which could be regained partially through connecting subunits in a trimer covalently using a disulfide bond. Gly mutation could have changed the tertiary structure of the protein to make it less compactly folded. Under this condition, potential trypsin digestion sites will be less protected than similar sites in a more compactly folded structure. Second, the mutation could have caused AcrB trimer to dissociate, under which condition the inter-subunit interface, which was protected in an AcrB trimer, would be exposed upon trimer dissociation. To characterize the tertiary structure of AcrBP223G, we used a disulfide-trapping Cycloheximide method developed earlier in our laboratory. We have previously identified seven pairs of residues in the periplasmic domain of AcrB that are within the disulfide bond distance. Mutations of these residues to Cys and formation of disulfide bonds at these positions have little effect on the function of AcrB. We have used these Cys-pair mutants as reporters to investigate the tertiary structure of AcrB, in which the formation of disulfide bond could be detected through labeling with a fluorescent probe. If the structure of a mutant is similar to that of WT AcrB, we expect to see a similar fluorescent labeling profile for the Cys pair reporters in both proteins.