Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Tuesday Speaker Abstracts

TOWARD UNDERSTANDING STRUCTURAL TRANSITIONS AND DYNAMICS IN PROTEINS USING PLASMON-ENHANCED SMFRET Mahran Shehade 1 ; Satyaghosh Maurya 1 ; Gilad Haran 1 ; 1 Weizmann Institute Of Science, Biological & Chemical Physics, Rehovot, Israel Proteins undergo dynamic conformational changes essential to their functions, and understanding these transitions at the single-molecule level has significant implications for biochemistry, bioengineering, and drug development. Transition paths (TPs), the transient trajectories through free-energy barriers, are key to understanding these changes. However, due to their fleeting nature, direct observation of TPs has been challenging. Advances in single-molecule FRET (smFRET) spectroscopy have provided insights into the average properties of TPs, but higher photon fluxes are needed to capture full trajectories at high temporal resolution. In this work, we harness Plasmonic Nanostructures, specifically zero-mode waveguides, to overcome the photon flux limitations that have long restricted smFRET. Guided by Finite-Difference Time-Domain and Boundary Element Method simulations, we tailor the geometry and materials of these nanostructures to maximize fluorophore excitation and emission rates, achieving photon counts of up to 10 million per second, approximately a 25-fold increase over conventional setups. This unprecedented photon flux enables direct, real-time detection of microsecond-scale conformational changes. Focusing on adenylate kinase, we apply this platform to capture TPs, yielding detailed distributions of TP times rather than merely average rates. This resolves fleeting, microsecond-scale events otherwise obscured by the time-averaging inherent to the lower photon flux and longer integration times of standard confocal methods. Preliminary experiments also explored how solvent viscosity affects TP measurements. By merging nanophotonic design with single-molecule spectroscopy, we demonstrate that tailored zero-mode waveguides provide the photon flux and time resolution needed to scrutinize rapid protein dynamics under near-physiological conditions. This enables future quantitative comparisons, such as applying Kramers-like or diffusion-barrier models to the observed TP distributions, while laying the groundwork for understanding fast structural transitions critical to protein function and regulation.

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