Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Poster Abstracts

61-POS Board 31 TOWARDS A MECHANISTIC UNDERSTANDING OF CONFORMATIONAL DYNAMICS AND LIGAND BINDING IN THE MULTI-DRUG ABC-EXPORTER BMRA USING SMFRET Anna-Katharina Spring 1,2 ; Alessandra Narducci 2 ; Veronika Osten 3 ; Kristin Oepen 3 ; Jean Michel Jault 4 ; Cédric Orelle 4 ; Dirk Schneider 3 ; Thorben Cordes 1,2 ; 1 Technische Universität Dortmund, Department of Chemistry and Chemical Biology, Dortmund, Germany 2 Ludwig-Maximilians-Universität München, Faculty of Biology, Munich, Germany 3 Johannes Gutenberg-Universität Mainz, Department Chemie, Mainz, Germany 4 CNRS - Institut de Biologie et Chimie des Protéines, Molecular Microbiology and Structural Biochemistry, Lyon, France ATP binding cassette transporters are one of the largest and oldest families of membrane transporters. With high relevance for the excretion of toxins as well as antibiotic- and drug resistance, especially human ABC transporters such as P-glycoprotein are considered clinically relevant due to their involvement in the excretion of drugs during chemotherapy. Structurally, all members of the ABC transporter family share a common basic architecture with two water soluble nucleotide binding domains (NBDs), powering active transport by ATP hydrolysis and two transmembrane domains (TMDs) forming the transport channel. The Bacillus multidrug resistance ATP-transporter, BmrA mediates the efflux of drugs frequently used in anti-cancer therapy. Although structures of BmrA in inward- and outward-facing conformation are available, the mechanisms of how the conformational changes are transmitted between the NBDs and the TMDs remain largely elusive. With the help of single-molecule Förster-resonance energy transfer (smFRET), we here aim to resolve conformational states and dynamical changes in BmrA. For assay design we first used the labelizer (https://labelizer.org) to select suitable cysteine variants of BmrA. We then verified if and how cysteine introduction and fluorophore labelling affect the ATPase activity of the protein. Using the best-performing variants, we started to visualize the conformational dynamics of BmrA upon ATP-binding and substrate transport, probing different domain motions of the protein. Results of conformational changes in the intracellular domain of the transporter confirm available structural and biochemical data of BmrA, where NBD closure is observed upon ATP addition. With the assay we aim to probe the timescales of conformational changes, the effects of transported substrates and those of the membrane mimicking environments (detergent vs. liposome).

93