Journal: Nature Communications

Article Title: Nanomechanical binding mechanism of ligands drives agonistic activity

doi: 10.1038/s41467-025-61929-1

Figure Lengend Snippet: a Schematic illustration of SMFS experiments, in which the unbinding forces between hIgG, coupled to the AFM tip via a PEG crosslinker, and the transmembrane hCD40 receptor were measured. b Exemplary force-distance curves from the SMFS experiments with distinct rupture events due to single (red) hIgG:hCD40 bond dissociation, and simultaneous (blue) or sequential (black) dissociation of two hIgG:hCD40 bonds. c Experimental pdfs (black curves) of unbinding forces for different contact times. Pdfs peaks were fitted with multi-Gaussians, representing unbinding forces of one (red) and two (blue) hIgG:CD40 bonds. The frequency of two bonds increased with longer contact times. d , e Increase of probability of hIgG binding P(t) and probability of second bond formation P 2 (t) over contact time, fitted using pseudo-first-order kinetics of a bimolecular reaction. f Unbinding force versus loading rate plot of hIgG2A. Data points (grey) were divided into loading rate segments. Mean and standard deviation of the first Gaussian (red dots) obtained from unbinding force pdfs of each segment were fitted using the Bell-Evans model (red fit). Higher forces were in good agreement with a Markov binding model (blue fit), predicting unbinding forces for parallel breakage of two identical bonds. Data presented are from three independent experiments.

Article Snippet: The cantilevers were then covered with a pre-mixed solution containing 100 μl of 1.5 μM His 6 -tagged human CD40 ligand (hCD40L; AcroBiosystems, CDL-H5248) and 4 μl of 5 mM NiCl 2 and stored overnight at 4 °C.

Techniques: Binding Assay, Standard Deviation