Browsing by Subject "ABC transporters"

Now showing 1 - 2 of 2
  • Research Data
    ATP-driven conformational dynamics reveal hidden intermediates in a heterodimeric ABC transporter
    2026-01-16
    Pečak, Matija
    Nocker, Christoph 
    Tampé, Robert 
    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.
      13  1
  • Research Data
    Single-molecule dynamics reveal ATP binding alone powers substrate translocation by an ABC transporter
    2025-07-25
    Christoph Nocker
    Pečak, Matija (DataCollector)
    Nocker, Tobias (DataCollector)
    Amin Fahim (DataCollector)
    Susac, Lukas (DataCollector)
    Tampé, Robert  (ContactPerson)
    ATP-binding cassette (ABC) transporters are molecular machines involved in diverse physiological processes, including antigen processing by TAP, a key component of adaptive immunity. TAP and its bacterial homolog TmrAB use ATP to translocate peptides across membranes, yet the precise mechanism linking ATP binding to substrate movement remains unclear. Here, we employ a single-molecule FRET sensor to visualize single translocation events by individual ABC transporters, overcoming the limitations of ensemble averaging. This approach reveals that substrate transport is driven by a conformational switch from the inward- to the outward-facing state. Using a slow-turnover TmrAB variant, we demonstrate that ATP binding alone, even in the absence of Mg2+, is sufficient to drive a single round of peptide translocation. Cryo-EM structures of wild-type and slow-turnover TmrAB show that ATP binding induces the outward-facing conformation without Mg2+. In wild-type TmrAB, this conformational transition supports a single translocation event, whereas Mg2+-dependent ATP hydrolysis is required to reset the transporter. These findings establish a direct mechanistic link between ATP binding and substrate translocation at single-molecule resolution, providing new insights into the catalytic cycle of ABC transporters.
      20  9