Fig. 1

HDX-MS reports on changes in the conformational equilibrium between IF and OF states. G to W mutants were used to highlight shifts in the conformational equilibrium. a Introduction of a tryptophan at the extracellular end of helix 2 sterically prevents closing. After deuteration, enzymatic digestion, and identification of the peptides by MS the mass uptake of the WT and mutants is compared. Peptides located on the intracellular side will be on average more deuterated than the peptides located on the extracellular side for the mutant (top graph) while the opposite pattern will be observed for the WT (bottom graph). b Differential deuterium uptake pattern (ΔHDX) mapped onto the 3D and topological structure of LacY (PDB: 2V8N), GlpT (PDB: 1PW4), and XylE (PDB: 4GBY). Red and blue colored regions indicate segments containing peptides with a positive ΔHDX (red—more deuteration) or negative ΔHDX (blue—less deuteration), respectively; white regions indicate that no significant ΔHDX is observed (P ≤ 0.01), and gray indicates regions where peptides were not obtained for both the mutant and the WT conditions. The yellow star indicates the location of the point mutation. All measurements were performed in triplicates. The ΔHDX datasets are presented as Woods plots in supplementary Figs.12 and 13