To import large metabolites across the outer membrane (OM) of Gram-negative bacteria, TonB dependent transporters (TBDTs) undergo significant conformational change. After substrate binding in BtuB, the E. coli vitamin B12 TBDT, TonB binds and couples BtuB to the inner membrane proton motive force which powers transport. The role of TonB in rearranging the plug domain to form a putative pore remains enigmatic. The aim of my thesis is to advance understanding of the substrate transport process in BtuB.Chapter 1 is a review of the outer membrane environment, TBDT biology, and a survey of what is known about BtuB specifically. Models of BtuB pore formation and some of the recent experiments supporting them are introduced. Some studies focus on force-mediated unfolding while others propose force-independent pore formation by TonB binding leading to breakage of a salt bridge termed the “Ionic Lock”. Additionally, an introduction is given to the technique of hydrogen deuterium exchange mass spectrometry (HDX-MS), with an emphasis on its application to outer membrane proteins. Chapter 2 details work characterizing early steps in the BtuBs transport cycle, in a native OM context. Our hydrogen exchange measurements in E. coli outer membranes find that a region surrounding the Ionic Lock, far from the B12 site, is fully destabilized upon substrate binding. A comparison of the exchange between the B12- and B12+TonB-bound complexes indicates that B12 binding is sufficient to unfold the Ionic Lock region with binding of a TonB fragment having much weaker effects. TonB binding accelerates exchange in the third substrate binding loop, but pore formation does not obviously occur. This chapter provides a detailed structural and energetic description of the early stages of B12 passage that provides support both for and against current models of the transport process. Chapter 3 presents a characterization of the independently folding plug domains found among a surveyed set of TBDTs. The plug domains of BtuB and CirA were found to contain substantial amounts of independently folding structure, and were characterized with a variety of techniques. Attempts at structure determination by NMR were unsuccessful due to poor solution properties and rapid transverse relaxation rates. Initial experiments characterizing BtuB constructs with stabilizing disulfide and/or bi-histidine site mutations were suspended upon success of initial mass spectrometry HDX experiments, which were used to characterize the folding of BtuB in a more native context. Chapter 4 summarizes the conclusions from chapter 2. From these conclusions, future directions are proposed to further advance understanding of BtuB biology, the mechanism of TBDTs in general, and to address an intriguing new question raised about apparent non-structural HDX protection conferred by the outer membrane. In summary, this thesis represents an advance in understanding the mechanism of BtuB and explores TBDT plug domain folding in various contexts. Additionally, it establishes a technique for routine acquisition of high-quality HDX data in a challenging but valuable context, the native outer membrane.