ATP-binding cassette (ABC) transporters are essential molecular machines whose conformational dynamics have largely been inferred from ensemble-averaged measurements. Resolving dynamic heterogeneity and transient intermediates, however, requires single-molecule approaches. Here, we use single-molecule Förster resonance energy transfer (smFRET) to directly monitor conformational changes of the heterodimeric type IV ABC transporter TmrAB, a functional homolog of the human antigen transporter TAP. Fluorophores positioned at the nucleotide-binding domains and the periplasmic gate were validated by accessible-volume simulations, fluorescence lifetimes, and ensemble FRET, demonstrating that these reporters reliably track conformational transitions. Single-molecule analysis distinguishes ATP-free and ATP-bound states and reveals ATP-dependent population shifts from micromolar to physiological ATP concentrations. Probing conformational dwell-times further uncovers an unexpectedly long ATP-bound dwell time of ~300 ms. Using complementary stabilization strategies–including a slow-turnover variant, Mg²⁺ depletion, or substrate trans-inhibition–we resolve a previously hidden outward-facing open state that rapidly interconverts with occluded intermediates under turnover conditions. These results provide the first single-molecule characterization of TmrAB and establish a general framework for dissecting ATP-coupled conformational dynamics in heterodimeric ABC transporters.

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