Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Tuesday Speaker Abstracts

EXPLORING DYNAMIC PROTEIN SYSTEMS WITH SINGLE-MOLECULE FRET Ecenaz Bilgen 1 ; Carina Fernandez Gonzalez 2 ; Léo Bonhomme 4 ; Mariam Mohamadi 3 ; Franz Hagn 3 ; Emmanuel Margeat 4 ; Robert B. Quast 4 ; Johannes Buchner 2 ; Don C. Lamb 1 ; 1 LMU Munich, Department of Chemistry and Center for Nanoscience, Munich, Germany 2 Technical University of Munich, Center for Functional Protein Assemblies (CPA), Department Bioscience, School of Natural Sciences, Munich, Germany 3 Technical University of Munich, Bavarian NMR Center (BNMRZ) and Structural Membrane Biochemistry, Dept. of Bioscience, TUM School of Natural Sciences, Munich, Germany 4 University of Montpellier, Centre de Biologie Structurale (CBS), CNRS, INSERM , Montpellier, France Single-molecule fluorescence resonance energy transfer (smFRET) has become a powerful approach for resolving the structure, dynamics, and interactions of biomolecules. This work presents a comprehensive investigation of dynamic protein systems using two- and three-color smFRET, with a particular focus on the solution-based multiparameter fluorescence detection with pulsed interleaved excitation (MFD-PIE) method. By combining fluorescence lifetime analysis, photon distribution analysis (PDA), and filtered fluorescence correlation spectroscopy (fFCS), conformational dynamics on the sub-millisecond timescale can be quantitatively characterized. The first part of this work investigates the conformational regulation of the tumor suppressor protein p53 by small heat shock proteins (sHsps). We show that α A- and α B crystallin (HspB4/HspB5) selectively bind p53 and trap it in a heterogeneous ensemble of non native conformations rather than a single defined state. We used FCS, PDA, and fluorescence lifetime analysis to elucidate the conformational changes occurring in the DNA-binding domain of p53 as it is released from the α B-crystallin complex. We demonstrate that p53 can be transferred to the ATP-dependent Hsp70/Hsp90 chaperone machinery for release and reactivation. The second part of this work demonstrates the application of two- and three-color smFRET to G protein-coupled receptor (GPCR) systems. By combining two-color smFRET with filtered fluorescence correlation spectroscopy (fFCS), we characterize the conformational changes and sub-millisecond dynamics of a transmembrane receptor. We further employ three color smFRET to investigate the human metabotropic glutamate receptor 2 (mGlu2), resolving coordinated conformational motions across multiple domains of the receptor. Together, these studies highlight the versatility of advanced smFRET methodologies for probing the complex conformational landscapes and dynamics of biologically important membrane proteins.

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