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

Abstract

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 bindinginduced 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. IMPORTANCEWe present a protocol for performing in vivo HDX-MS, focusing on BtuB, a protein whose native membrane environment is believed to be mechanistically

Folding of Prestin’s Anion-Binding Site and the Mechanism of Outer Hair Cell Electromotility

“…Eluted peptides were analyzed by a Thermo Q Exactive mass spectrometer. MS data collection, peptide assignments by SearchGUI version 4.0.25, and HDX data processing by HDExaminer 3.1 (Sierra Analytics) were performed as previously described 22,23 .…”

“…than in bulk solvent 25,26 . For prestin, we propose that detergent molecules in the micelle can restrict the access of ODto amide protons, leading to a local effective pD lower than the bulk solvent and hence producing the apparent PF for the unfolded N-terminus of TM3.…”

Hydrogen/deuterium exchange-mass spectrometry of integral membrane proteins in native-like environments: current scenario and the way forward

“…The Sosnick group developed a protocol for in vivo HDX-MS using the model IMP BtuB ( Figure 3 ) [ 61 ]. The protein was labelled in live E. coli cells by transferring them to deuterated growth media.…”

“… Figure taken from Lin et al [ 61 ] with permission from the publisher (Protein Science). ( A ) For in vivo HDX-MS, living E. coli cells overexpressing the protein of interest (in this case BtuB) are diluted in D 2 O buffer supplemented with a carbon source.…”

Structural mass spectrometry approaches to understand multidrug efflux systems

“…By analysing the deuterium uptake of OmpF in its native environment, they were able to report areas of the protein that were buried or exposed, which was in good agreement with previous X-ray diffraction data. Recent work by the Sosnick group has studied the TonB-dependant transporter, BtuB, using HDX-MS with increasing complexity [ 86 , 87 ]. One method was measuring BtuB structural dynamics within the E. coli outer membrane, similar to the study by Donnarumma et al The second method used a protocol developed for in vivo HDX-MS. By over expressing cells with BtuB and diluting into deuterated LB buffer, they achieved desired deuterium labelling.…”