ArticleProtein science : a publication of the Protein Society2022
Development of in vivo HDX-MS with applications to a TonB-dependent transporter and other proteins.
Article in Protein science : a publication of the Protein Society, 2022. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 11 papers.
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Who cites it
11 citing papers in PubMed, 21 citations in OpenAlex.
- Probing protein-lipid interactions by hydrogen/deuterium exchange-mass spectrometry: advances, challenges and future directions.Chemical science · 2026Review
- Structural dynamics and immunogenicity of the recombinant and outer membrane vesicle-embedded Meningococcal antigen NadA.Nature communications · 2026Article
- Epitope Screening by Hydrogen-Deuterium Exchange Mass Spectrometry for Improved Immunotherapeutic Outcomes.Methods in molecular biology (Clifton, N.J.) · 2026Article
- Practical Tips for the Application of HDX-MS to Membrane Proteins, Biomolecular Condensates, and Weak Protein Binders.Journal of the American Society for Mass Spectrometry · 2025Article
- Target Engagement Assays in Early Drug Discovery.Journal of medicinal chemistry · 2025Review
- Article
- Mass Spectrometry Investigation of Some ATP-Binding Cassette (ABC) Proteins.Medicina (Kaunas, Lithuania) · 2024Review
- Folding of prestin's anion-binding site and the mechanism of outer hair cell electromotility.eLife · 2023Article
- Hydrogen/deuterium exchange-mass spectrometry of integral membrane proteins in native-like environments: current scenario and the way forward.Essays in biochemistry · 2023Article
- Structural mass spectrometry approaches to understand multidrug efflux systems.Essays in biochemistry · 2023Review
- Development of in vivo HDX-MS with applications to a TonB-dependent transporter and other proteins.Protein science : a publication of the Protein Society · 2022Article
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Authors and funding
5 authors at 2 institutions in 1 country.
Funding
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 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.
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