Ligand Translational Mobility and Concentration Near Membranes

该项目的假设是，局部偏离理想的，大量的翻译迁移率和浓度的配体膜表面附近的配体施加关键影响的配体-受体相互作用的动力学，因此，生物功能。 为了检验这一假设，将追求三组具体目标。 在第一组的目标，物理因素（静电，流体动力学和截留）管理的翻译迁移率和浓度的荧光配体的区域的解决方案，是非常接近的模型和自然细胞膜沉积在透明的平面基板将被检查。 这些测量将使用上一个资助期间开发的全内反射荧光相关光谱法进行。 在第二组具体目标中，将实施三项技术创新。 这些创新将提供依赖于距离，而不是距离平均，当地配体动力学的信息。 首先，通过改变倏逝波深度来探测局部配体平移迁移率和浓度的距离依赖性。第二，光子计数直方图与倏逝照明获得将提供一个直接测量的方式，其中的配体浓度，因此潜在的依赖于距离膜表面的距离。第三，高阶自相关将被用来进一步确认和理解附近的膜表面上的局部配体浓度和迁移率的影响。 最终的具体目标是将所观察到的配体浓度和动力学的局部变化与特定的配体-受体动力学相关联。 相互作用动力学将采用完善的方法（全内反射与荧光光漂白恢复）进行监测。

The hypothesis of this project is that local deviations from ideal, bulk translational mobilities and concentrations of ligands near membrane surfaces exert critical influences on the kinetics of ligand-receptor interactions and, as a consequence, biological function. To test this hypothesis, three groups of specific aims will be pursued. In the first group of aims, physical factors (electrostatic, hydrodynamic and entrapment) governing the translational mobilities and concentrations of fluorescent ligands in regions of solution that are very close to model and natural cell membranes deposited on transparent planar substrates will be examined. These measurements will be carried out by using total internal reflection with fluorescence correlation spectroscopy as developed during the last funding period. In the second group of specific aims, three technological innovations will be implemented. These innovations will provide distance-dependent, rather than distance-averaged, information about local ligand dynamics. First, the distance-dependence of the local ligand translational mobility and concentration will be probed by changing the evanescent depth. Second, photon counting histograms obtained with evanescent illumination will provide a direct measure of the manner in which the ligand concentration and therefore potential depend on the distance from the membrane surface. Third, high order autocorrelation will be employed to further confirm and understand the effects of the nearby membrane surface on the local ligand concentration and mobility. The goal of the final specific aim is to correlate local changes in ligand concentrations and dynamics, as they are observed, with specific ligand-receptor kinetics. The interaction kinetics will be monitored with a well-established method, total internal reflection with fluorescence photobleaching recovery.