Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland 21218, United States.
Langmuir. 2013 Feb 19;29(7):2299-310. doi: 10.1021/la304355t. Epub 2013 Feb 4.
We present diffusing colloidal probe measurements of weak, multivalent, specific protein-polysaccharide interactions mediated by a competing monosaccharide. Specifically, we used integrated evanescent wave and video microscopy methods to monitor the three-dimensional Brownian excursions of conconavilin A (ConA) decorated colloids interacting with dextran-functionalized surfaces in the presence of glucose. Particle trajectories were interpreted as binding lifetime histograms, binding isotherms, and potentials of mean force. Binding lifetimes and isotherms showed clear trends of decreasing ConA-dextran-specific binding with increasing glucose concentration, consistent with expectations. Net potentials were accurately captured by superposition of a short-range, glucose-independent ConA-dextran repulsion and a longer-range, glucose-dependent dextran bridging attraction modeled as a harmonic potential. For glucose concentrations greater than 100 mM, the net ConA-dextran potential was found to have only a nonspecific repulsion, similar to that of bovine serum albumin (BSA) decorated colloids over dextran determined in control experiments. Our results demonstrate the first use of optical microscopy methods to quantify the connections between potentials of mean force and the binding behavior of ConA-decorated colloids on dextran-functionalized surfaces.
我们展示了胶体探针的扩散测量结果，这些结果涉及由竞争单糖介导的弱多价特定蛋白质-多糖相互作用。具体来说，我们使用集成的消逝波和视频显微镜方法来监测在葡萄糖存在下，与葡聚糖功能化表面相互作用的 ConA 修饰胶体的三维布朗运动。颗粒轨迹被解释为结合寿命直方图、结合等温线和平均力势。结合寿命和等温线显示出随着葡萄糖浓度增加，ConA-葡聚糖特异性结合明显减少的趋势，这与预期一致。通过将短程、与葡萄糖无关的 ConA-葡聚糖排斥和长程、与葡萄糖相关的葡聚糖桥接吸引叠加，可以准确地捕获净位势，后者被建模为调和位势。对于葡萄糖浓度大于 100 mM 的情况，发现净 ConA-葡聚糖位势仅具有非特异性排斥作用，类似于在对照实验中确定的在葡聚糖上修饰胶体的牛血清白蛋白 (BSA) 的排斥作用。我们的结果首次证明了使用光学显微镜方法来定量平均力势与 ConA 修饰胶体在葡聚糖功能化表面上的结合行为之间的联系。
