Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Wednesday Speaker Abstracts

CHARACTERIZING ULTRAFAST BARRIER CROSSING DYNAMICS OF PROTEIN FOLDING USING TWO- AND THREE-COLOR SINGLE-MOLECULE FRET IN ZERO-MODE WAVEGUIDES Chi-Jui Feng 1 ; Hoi Sung Chung 1 ; 1 National Institutes of Health, Laboratory of Chemical Physics, Bethesda, MD, USA Transition paths (TPs) describe short-lived, intrinsically heterogeneous, single-molecule barrier crossing events that contain all the mechanistic details of molecular transitions such as folding and binding of proteins and nucleic acids. Single-molecule spectroscopy has been used to probe TPs including determining TP times on the order of 10–100 μ s and characterizing free energy landscape along one-dimensional coordinate and diversity of TPs. However, the limited fluorescence brightness in single-molecule FRET impedes detailed characterization of the protein-folding TPs on the order of 1 μ s or even shorter. This can be overcome by using zero mode waveguides (ZMWs) to enhance fluorescence brightness. We combined single-molecule FRET spectroscopy with ZMWs to study protein folding TPs. Waveguides successfully enhanced the brightness to several MHz, enabling the study of microsecond TPs. We surveyed eight naturally evolved single-domain proteins with varied sizes, secondary structures, and folding rates to examine the relations among various physicochemical properties, kinetics, and barrier crossing dynamics. We found that the TP times are short (0.7 - 4 μ s) and insensitive to these properties, but barrier curvature, modulated by the barrier height, is a strong determinant of TP times. Surprisingly, the diffusion coefficient across the free energy barrier, extracted from the TP times, increases with protein size, suggesting greater cooperativity of native contact formation in larger proteins. Our results reveal that evolutionary optimization of the energy landscape is much more efficient for protein folding than for other biomolecular processes. We extended our study to probe the conformational details of heterogeneous TPs of a designed three helix bundle protein using three-color single-molecule FRET. We observed two clusters of TPs. In both TP clusters, the two C-terminal helices are partially folded, whereas the disordered N terminal helix is either extended or collapsed.

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