ABSTRACTThe specific binding of ligands by proteins and the coupling of this process to conformational changes are fundamental to protein function. We designed a fluorescence-based single-molecule assay and data analysis procedure that allows the simultaneous real-time observation of ligand binding and conformational changes in FeuA. The substrate-binding protein FeuA binds the ligand ferri-bacillibactin and delivers it to the ABC importer FeuBC, which is involved in iron uptake in bacteria. The conformational dynamics of FeuA was assessed via Förster resonance energy transfer (FRET), whereas the presence of the ligand was probed by fluorophore quenching. We reveal that ligand binding shifts the conformational equilibrium of FeuA from an open to a closed conformation. Ligand binding occurs via an induced-fit mechanism, i.e., the ligand binds to the open state and subsequently triggers a rapid closing of the protein. However, FeuA also rarely samples the closed conformation without the involvement of the ligand. This shows that ligand interactions are not required for conformational changes in FeuA. However, ligand interactions accelerate the conformational change 10000-fold and temporally stabilize the formed conformation 250-fold.SIGNIFICANCE STATEMENTLigand binding and the coupling of this process to conformational changes in proteins are fundamental to their function. We developed a single-molecule approach that allows the simultaneous observation of ligand binding and conformational changes in the substrate-binding protein FeuA. This allows to directly observe the ligand binding process, ligand-driven conformational changes as well as rare short-lived conformational transitions that are uncoupled from the ligand. These findings provide insight into the fundamental relation between ligand-protein interactions and conformational changes. Our findings are, however, not only of interest to understand protein function, but the developed data analysis procedure allows the determination of (relative) distance changes in single-molecule FRET experiments, for situations in which donor and acceptor fluorophore are influenced by quenching processes.
Photovoice Data Analysis: Critical Approach, Phenomenological Approach, and Beyond