Regensburg 2025 – scientific programme
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BP: Fachverband Biologische Physik
BP 7: Single Molecule Biophysics
BP 7.3: Talk
Monday, March 17, 2025, 15:45–16:00, H44
Two-color single-molecule coincidence detection for the analysis of biological processes and high-affinity bi-molecular binding — •Benno Schedler1, Olessya Yuknovets1, Alida Meyer1, Lennart Lindner1, and Jörg Fitter1, 2 — 1RWTH Aachen University, I. Physikalisches Institut (IA), Aachen, Germany — 2FZ Jülich, ER-C-3, Jülich, Germany
Life on the molecular scale is based on a versatile interplay of biomolecules, a feature that is relevant for the formation of macromolecular complexes. Fluorescence based two-color coincidence detection is widely used to characterize molecular binding and was recently improved by a brightness-gated version which gives more accurate results [1]. We developed and established protocols which make use of coincidence detection to quantify binding fractions between interaction partners labeled with fluorescence dyes of different colors. Since the applied technique is intrinsically related to single molecule detection, the concentration of diffusing molecules for confocal detection is typically in the low pico-molar regime. This makes the approach a powerful tool for determining bi-molecular binding affinities, in terms of KD-values, in this regime. By measuring the affinity at different temperatures, we were able to determine thermodynamic parameters of the binding interaction. The results show that the ultra-tight binding is dominated by entropic contributions [2].
References:
[1] Höfig et al. Communication Biology 2019 2, 459
[2] Schedler et.al. Int. J. Mol.Sci 2023 24, 16379
Keywords: Confocal Microscopy; bi-molecular binding; TCCD; single molecule