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BP: Fachverband Biologische Physik

BP 7: Single Molecule Biophysics

BP 7.3: Vortrag

Montag, 17. März 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 Fitter^{1, 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