Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Poster Abstracts

35-POS Board 17 FRETNET DESIGNER ENABLES RATIONAL SMFRET MAPPING OF RIBOSE BINDING PROTEIN CONFORMATIONAL STATES AND DYNAMICS George Hamilton 1,2 ; Aishwarya Krishnamohan 3 ; Francis Duffy 1,4 ; Sanjeev Ghimire 5 ; Anay Lazaro-Alfaro 1 ; Jason Martin 1 ; Faruck Morcos 3 ; Hugo Sanabria 1,5 ; 1 Clemson University, Department of Physics and Astronomy, Clemson, SC, USA 2 Inizio Medical, Warminster, PA, USA 3 University of Texas at Dallas, Departments of Biological Sciences and Bioengineering, Dallas, TX, USA 4 Novonesis, Franklinton, NC, USA 5 Clemson University, Medical Biophysics Program, Clemson, SC, USA The multidomain D-ribose-binding protein (RbsB) is a periplasmic component of ATP-binding cassette (ABC) transport systems responsible for bacterial sugar uptake and chemotactic signaling. Classically, its conformational landscape has been described by two states: an open apo conformation and a closed ribose-bound conformation. However, recent direct coupling analysis (DCA) combined with structure-based modeling predicted a third twisted intermediate state, equidistant between the canonical conformations, suggesting previously unrecognized functional complexity. Here, we employ single-molecule FRET (smFRET) to experimentally resolve the RbsB conformational ensemble and characterize its dynamic interconversion. To rationally identify labeling positions, we developed FRETnet Designer, a computational pipeline integrating structure- and sequence-based optimization for FRET network construction. Using this framework, we determined that at least three double-labeled RbsB variants are required to resolve the predicted conformational landscape. We expressed, purified, and site-specifically labeled the selected variants and subsequently characterized them by multiparameter fluorescence detection (MFD) in a confocal microscope operating in pulse-interleaved excitation mode with four-channel polarized, time-tagged, time-resolved single-photon detection. Time correlated single-photon counting (TCSPC) combined with photon distribution analysis (PDA) revealed substantial conformational heterogeneity in apo RbsB across variants. Titration with ribose progressively altered FRET efficiencies and shifted two-dimensional histograms of corrected FRET efficiency versus donor lifetime toward a static FRET regime, indicating stabilization of a less dynamic, ligand-bound conformational ensemble. Collectively, these findings provide experimental evidence for a complex, multidimensional conformational landscape in RbsB and establish FRETnet Designer as a robust strategy for minimally perturbative fluorophore placement and high-resolution characterization of protein structure and dynamics by smFRET.

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