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Biophys J. 2019 Nov 5;117(9):1642-1654. doi: 10.1016/j.bpj.2019.08.005. Epub 2019 Aug 12.

Single-Molecule Observation of Ligand Binding and Conformational Changes in FeuA.

Author information

1
Molecular Microscopy Research Group, Zernike Institute for Advanced Materials, University of Groningen, Groningen, the Netherlands.
2
Molecular Microscopy Research Group, Zernike Institute for Advanced Materials, University of Groningen, Groningen, the Netherlands; Physical and Synthetic Biology, Faculty of Biology, Ludwig-Maximilians-Universität München, Planegg-Martinsried, Germany; KU Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Laboratory of Molecular Bacteriology, Leuven, Belgium.
3
Molecular Microscopy Research Group, Zernike Institute for Advanced Materials, University of Groningen, Groningen, the Netherlands; Physical and Synthetic Biology, Faculty of Biology, Ludwig-Maximilians-Universität München, Planegg-Martinsried, Germany. Electronic address: cordes@bio.lmu.de.

Abstract

The specific binding of ligands by proteins and the coupling of this process to conformational changes is 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 ATP-binding cassette importer FeuBC, which is involved in bacterial iron uptake. The conformational dynamics of FeuA was assessed via Förster resonance energy transfer, 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 10,000-fold and temporally stabilize the formed conformation 250-fold.

PMID:
31537314
PMCID:
PMC6838762
[Available on 2020-11-05]
DOI:
10.1016/j.bpj.2019.08.005
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