Single-molecule observation of ligand binding and conformational changes in FeuA (Biophysical Journal)

The 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 bacterial iron uptake. The conformational dynamics of FeuA was assessed via Förster resonance energy transfer (FRET), whereas the presence of the ligand was probed by fluorophore quenching.