Minimal encounter time and separation determine ligand-receptor binding in cell adhesion.

The binding properties of biomolecules play a crucial role in many biological phenomena, especially cell adhesion. Whereas the attachment kinetics of soluble proteins is considered well known, complex behavior arises when protein molecules are bound to the cell membrane. We probe the hidden kinetics of ligand-receptor bond formation using single-molecule flow chamber assays and Brownian dynamics simulations. We show that, consistent with our recently proposed hypothesis, association requires a minimum duration of contact between the reactive species. In our experiments, ICAM-1 anchored on a flat substrate binds to anti-ICAM-1 coated onto flowing microbeads. The interaction potential between bead and substrate is measured by microinterferometry and is used as an ingredient to simulate bead movement. Our simulation calculates the duration of ligand-receptor contacts imposed by the bead movement. We quantitatively predict the reduction of adhesion probability measured for shorter tether length of the ligand or if a repulsive hyaluronan layer is added onto the surface. To account for our results, we propose that bond formation may occur in our system by crossing of a diffusive plateau in the energy landscape, on the timescale of 5 ms and an energy barrier of 5 k(B)T, before reaching the first detectable bound state. Our results show how to relate cell-scale behavior to the combined information of molecular reactivity and biomolecule submicron-scale environment.