We found a match
Your institution may have access to this item. Find your institution then sign in to continue.
- Title
The Thermodynamic Fingerprints of Ultra-Tight Nanobody–Antigen Binding Probed via Two-Color Single-Molecule Coincidence Detection.
- Authors
Schedler, Benno; Yukhnovets, Olessya; Lindner, Lennart; Meyer, Alida; Fitter, Jörg
- Abstract
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. 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 picomolar regime. This makes the approach a powerful tool for determining bi-molecular binding affinities, in terms of KD values, in this regime. We demonstrated the reliability of our approach by analyzing very strong nanobody-EGFP binding. By measuring the affinity at different temperatures, we were able to determine the thermodynamic parameters of the binding interaction. The results show that the ultra-tight binding is dominated by entropic contributions.
- Subjects
COINCIDENCE circuits; CONFOCAL fluorescence microscopy
- Publication
International Journal of Molecular Sciences, 2023, Vol 24, Issue 22, p16379
- ISSN
1661-6596
- Publication type
Article
- DOI
10.3390/ijms242216379