Mapping of RNA Helicase Dynamics by HDX-MS.

RNA helicases are RNA-binding enzymes that remodel RNA structures and ribonucleoprotein complexes through regulated conformational changes. Their RecA-like catalytic cores cooperate with auxiliary domains, many of which contain flexible or partially disordered regions whose rearrangements are difficult to characterize using static structural methods. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) overcomes this limitation by examining protein conformation in solution, providing peptide-resolved information on local stability, dynamics, and ligand-dependent structural changes. In this chapter, we describe a bottom-up HDX-MS workflow for mapping RNA helicase-RNA interactions and conformational dynamics, using the Pseudomonas aeruginosa DExH-box helicase HrpB as a model. The protocol covers sample preparation, deuterium labeling, quenching, protein digestion, peptide separation, MS acquisition, and data analysis. We outline practical guidelines for optimizing peptide coverage, preserving deuterium labeling throughout the workflow, and obtaining quantitative comparisons between the apo and RNA-bound states. The chapter provides a framework for using HDX-MS to identify where RNA, nucleotides, and small-molecule inhibitors bind, to characterize the conformational changes they induce, and to connect these dynamics to the RNA helicase mechanism of action.