Development of in vivo HDX-MS with applications to a TonB-dependent transporter and other proteins

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a powerful tool that monitors protein dynamics in solution. However, the reversible nature of HDX labels has largely limited the application to in vitro systems. Here, we describe a protocol for measuring HDX-MS in living Escherichia coli cells applied to BtuB, a TonB-dependent transporter found in outer membranes (OMs). BtuB is a convenient and biologically interesting system for testing in vivo HDX-MS due to its controllable HDX behavior and large structural rearrangements that occur during the B12 transport cycle. Our previous HDX-MS study in native OMs provided evidence for B12 binding and breaking of a salt bridge termed the Ionic Lock, an event that leads to the unfolding of the amino terminus. Although purified OMs provide a more native-like environment than reconstituted systems, disruption of the cell envelope during lysis perturbs the linkage between BtuB and the TonB complex that drives B12 transport. The in vivo HDX response of BtuB's plug domain (BtuBp) to B12 binding corroborates our previous in vitro findings that B12 alone is sufficient to break the Ionic Lock. In addition, we still find no evidence of B12 binding-induced unfolding in other regions of BtuBp that could enable B12 passage. Our protocol was successful in reporting on the HDX of several endogenous E. coli proteins measured in the same measurement. Our success in performing HDX in live cells opens the possibility for future HDX-MS studies in a native cellular environment. IMPORTANCE We present a protocol for performing in vivo HDX-MS, focusing on BtuB, a protein whose native membrane environment is believed to be mechanistically important for B12 transport. The in vivo HDX-MS data corroborate the conclusions from our previous in vitro HDX-MS study of the allostery initiated by B12 binding. Our success with BtuB and other proteins opens the possibility for performing additional HDX-MS studies in a native cellular environment.