Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Tuesday Speaker Abstracts

THE SPEED LIMIT OF GENOMIC SEARCH: A KINETIC RACE BETWEEN ELECTROSTATIC STEERING, SLIDING FRICTION, AND CONFORMATIONAL LOCKING Catherine Ghosh 1,2 ; Rama R Goluguri 3 ; Mourad Sadqi 2 ; Victor Muñoz 2 ; 1 University of Copenhagen, Department of Biology, Copenhagen, Denmark 2 University of California Merced, Department of Bioengineering, Merced, CA, USA 3 Stanford University, Department of Biochemistry, Palo Alto, CA, USA Transcription factors (TFs) navigate the vast eukaryotic genome to locate specific target sites with remarkable speed and precision. While facilitated diffusion via 1D sliding is a widely accepted search mechanism, the precise physical principles governing the transition from rapid scanning to stable recognition remain unresolved. Specifically, the interplay between long-range electrostatic steering, the "friction" of sliding, and the conformational changes required for specific binding is poorly understood. In this work, we resolve this search mechanism for the Engrailed homeodomain (enHD) using advanced single-molecule FRET (smFRET) spectroscopy and Maximum Likelihood Analysis of Photon Arrival Times (MLA-PAT). By dissecting the binding kinetics across a broad range of ionic strengths and DNA lengths, we successfully extricated the distinct contributions of bulk electrostatics, local DNA field effects, and 1D sliding dynamics. Our results reveal that the search process is a finely tuned kinetic race. We experimentally determined the Debye length of the DNA electric field (0.82 nm at physiological ionic strength), confirming that electrostatic steering accelerates the initial capture. However, we uncovered a critical "sliding bottleneck": while longer DNA antennas enhance protein recruitment, the time cost of sliding eventually hampers the specific "lock-into-target" (cognate site binding) step. Mechanistically, we show that specific recognition is not merely diffusion arrest but is gated by a rate-limiting conformational switch. Crucially, we find that the DNA’s local electric field acts as an active catalyst by accelerating the protein's conformation switch. This switch acts as a "molecular brake," rheostatically tuned by the protein’s stability to halt diffusion precisely at the cognate site. This establishes a quantitative framework for understanding how eukaryotic TFs optimize the trade-off between search speed and recognition fidelity, avoiding the "stickiness" of non-specific traps while ensuring reliable gene activation.

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