Connecting Protein Conformation and Dynamics with Ligand–Receptor Binding Using Three-Color Förster Resonance Energy Transfer Tracking

Specific binding
between biomolecules, i.e., molecular recognition, controls virtually
all biological processes including the interactions between cells
and biointerfaces, both natural and synthetic. Such binding often
relies on the conformation of biomacromolecules, which can be highly
heterogeneous and sensitive to environmental perturbations, and therefore
difficult to characterize and control. An approach is demonstrated
here that directly connects the binding kinetics and stability of
the protein receptor integrin αvβ3 to the conformation of the ligand fibronectin (FN), which are believed
to control cellular mechanosensing. Specifically, we investigated
the influence of surface-adsorbed FN structure and dynamics on αvβ3 binding using high-throughput single-molecule
three-color Förster resonance energy transfer (FRET) tracking
methods. By controlling FN structure and dynamics through tuning surface
chemistry, we found that as the conformational and translational dynamics
of FN increased, the rate of binding, particularly to folded FN, and
stability of the bound FN−αvβ3 complex decreased significantly. These findings highlight the importance
of the conformational plasticity and accessibility of the arginine-glycine-aspartic
acid (RGD) binding site in FN, which, in turn, mediates cell signaling
in physiological and synthetic environments.