TY  - JOUR
AU  - Schedler, Benno
AU  - Yukhnovets, Olessya
AU  - Lindner, Lennart
AU  - Meyer, Alida
AU  - Fitter, Jörg
TI  - The Thermodynamic Fingerprints of Ultra-Tight Nanobody–Antigen Binding Probed via Two-Color Single-Molecule Coincidence Detection
JO  - International journal of molecular sciences
VL  - 24
IS  - 22
SN  - 1422-0067
CY  - Basel
PB  - Molecular Diversity Preservation International
M1  - FZJ-2023-05361
SP  - 16379 -
PY  - 2023
AB  - Life on the molecular scale is based on a versatile interplay of biomolecules, a featurethat is relevant for the formation of macromolecular complexes. Fluorescence-based two-colorcoincidence detection is widely used to characterize molecular binding and was recently improvedby a brightness-gated version which gives more accurate results. We developed and establishedprotocols which make use of coincidence detection to quantify binding fractions between interactionpartners labeled with fluorescence dyes of different colors. Since the applied technique is intrinsicallyrelated to single-molecule detection, the concentration of diffusing molecules for confocal detectionis typically in the low picomolar regime. This makes the approach a powerful tool for determiningbi-molecular binding affinities, in terms of KD values, in this regime. We demonstrated the reliabilityof our approach by analyzing very strong nanobody-EGFP binding. By measuring the affinity atdifferent temperatures, we were able to determine the thermodynamic parameters of the bindinginteraction. The results show that the ultra-tight binding is dominated by entropic contributions.
LB  - PUB:(DE-HGF)16
C6  - 38003569
UR  - <Go to ISI:>//WOS:001113898000001
DO  - DOI:10.3390/ijms242216379
UR  - https://juser.fz-juelich.de/record/1019401
ER  -