Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Wednesday Speaker Abstracts

DIRECT QUANTIFICATION OF PROTEIN-PROTEIN INTERACTIONS IN LIVING BACTERIAL CELLS BY ACCURATE FRET MEASUREMENT Nam Ki Lee 1 ; 1 Seoul National University, Chemistry, Seoul, South Korea Quantitative measurement of protein–protein interactions (PPIs) within living cells is essential for understanding cellular functions at the molecular level and for advancing applications in synthetic biology, protein engineering, and drug discovery. Although numerous methods have been developed to quantify PPI strength in vitro, directly measuring PPIs inside living bacterial cells remains highly challenging. Here, we report KD-FRET, a fluorescence resonance energy transfer (FRET)-based method that quantitatively determines dissociation constants (KdK_dKd) of PPIs in living E. coli cells. We found that direct excitation of the acceptor fluorophore arising from spectral crosstalk can cause noninteracting protein pairs to exhibit an apparent KdK_dKd, thereby generating false-positive signals. KD-FRET effectively quantifies a wide range of PPI affinities, including both heterologous and homologous interaction pairs. Importantly, the method also enables measurement of KdK_dKd values for interaction pairs that cannot be characterized in vitro because of their instability under standard buffer conditions. Furthermore, KD-FRET was successfully applied to engineer metabolic pathways for enhanced naringenin production in E. coli and lycopene production in S. cerevisiae, demonstrating its utility as a synthetic biology platform. These results establish KD-FRET as a powerful and versatile approach for quantitatively studying PPIs in their native cellular environments. UNCOVERING NOVEL G-PROTEIN PATHWAYS USING LIVE CELL IMAGING Greta E. Schmidt ; Madison R Rennie 1 ; Lela Jackson 1 ; Androniqi Qifti 1 ; Suzanne F Scarlata 1 ; 1 Worcester Polytechnic Institute, Department of Chemistry & Biochemistry, Worcester, MA, USA Many common pharmaceuticals work through the G α q/PLC signaling system for their therapeutic benefits. When these drugs bind to their receptors, they activate G α q which then activates PLC, producing calcium signals in cells. Previous work by our lab has quantified the interaction between the components of the G α q/PLC pathway in model membranes. When studied in cells, we see cellular behavior which is independent of calcium and G α q. Unexpectedly, we find that PLC has a cytosolic population that affects protein translation and microRNA populations. Using FRET and single cell imaging, we find that PLC inhibits the promoter of RNA induced silencing (RISC) to change the microRNA population. Additionally, PLC binds to a component of RISC which forms stress granules. Because of these interactions, changes in cytosolic PLC levels impact protein translation which changes the differentiation state of cells. We follow these changes in cultured neuronal cells, muscle cells, and in the small organism C. elegans.

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