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.

生物分子间的特异性结合，即分子识别，几乎调控了所有生命过程，涵盖细胞与天然、合成生物界面之间的相互作用。此类结合通常依赖于生物大分子的构象，而生物大分子构象往往具有高度异质性且对环境扰动敏感，因此难以表征与调控。本研究展示了一种可直接将蛋白质受体整合素αvβ3（integrin αvβ3）的结合动力学与结合稳定性，与配体纤连蛋白（fibronectin, FN）的构象相关联的方法，而二者被认为是调控细胞机械感知的关键因素。具体而言，本研究借助高通量单分子三色荧光共振能量转移（Förster resonance energy transfer, FRET）追踪技术，探究了表面吸附纤连蛋白的结构与动力学行为对整合素αvβ3结合过程的影响。通过调控表面化学性质来控制纤连蛋白的结构与动力学行为，研究发现随着纤连蛋白构象与平移动力学的增强，其结合速率（尤其是与折叠态纤连蛋白的结合速率）以及结合态纤连蛋白-整合素αvβ3复合物的稳定性均显著下降。本研究结果凸显了纤连蛋白中精氨酸-甘氨酸-天冬氨酸（arginine-glycine-aspartic acid, RGD）结合位点的构象可塑性与可及性的重要性，而该位点可进一步介导生理及合成环境中的细胞信号转导。