Publication

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

de Boer, M., Gouridis, G., Muthahari, Y. A. & Cordes, T., 5-Nov-2019, In : Biophysical Journal. 117, 9, p. 1642-1654 13 p.

Research output: Contribution to journalArticleAcademicpeer-review

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 Forster 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.

Original languageEnglish
Pages (from-to)1642-1654
Number of pages13
JournalBiophysical Journal
Volume117
Issue number9
Publication statusPublished - 5-Nov-2019

    Keywords

  • RESONANCE ENERGY-TRANSFER, PROTEIN, DYNAMICS, TRANSPORTERS, TRANSITIONS, INSIGHTS, RECEPTOR, MOTIONS, FRET

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