In E. coli, BtuB, which is located in the OM, transports vitamin B12 (VitB12) into the periplasm through the interaction with TonB and the ExbB-ExbD complex. Subsequently, the periplasmic binding protein BtuF binds and delivers VitB12 to BtuCD, which transports it across the IM at the expense of ATP binding and hydrolysis (Figure ). The two BtuC subunits, which are inserted into the IM and connected with two solvent-exposed BtuD subunits (which bind and hydrolyze ATP), form an intact We isolated the cellular envelope following overexpression of BtuCD in E. coli. A cysteine substitution was introduced on the short loop between the transmembrane helices 5 and 5a (TM5 and TM5a) at T168 in the BtuC subunits (Figure and). The expression level for BtuCD was optimized by testing various growth conditions, and its presence in the native envelope was further confirmed using Western blotting (Figure ). Previously, we investigated these positions in micelles, and this variant demonstrated ATPase activity comparable to the wild-type. We labeled these cysteines directly in the envelope by incubation with the nitroxide label S- (1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl)methyl methanesulfonothioate (MTSL, please see the methods section). The MTSL label is stable in the envelope environment for a sufficiently long time (observed up to ~2 h, (Figure ).