Ligand Translational Mobility and Concentration Near Membranes

这个项目的假设是，局部偏离理想的整体平移迁移率和膜表面附近配体的浓度对配体-受体相互作用的动力学产生关键影响，从而影响生物功能。为了验证这一假设，我们将追求三组具体目标。在第一组AIMS中，将研究控制荧光配体在非常接近模型和沉积在透明平面底物上的天然细胞膜的溶液区域中的平移迁移率和浓度的物理因素(静电、流体动力学和捕获)。这些测量将使用上一次供资期间开发的全内反射和荧光相关光谱学进行。在第二组具体目标中，将实施三项技术创新。这些创新将提供与距离相关的、而不是距离平均的关于局部配体动力学的信息。首先，通过改变消逝深度来研究局部配体的平移迁移率和浓度与距离的关系。其次，用消逝光获得的光子计数直方图将提供配体浓度和电势依赖于离膜表面距离的直接测量方式。第三，利用高阶自相关进一步确认和理解附近膜表面对局部配体浓度和迁移率的影响。最终特定目标的目标是将观察到的配体浓度和动力学的局部变化与特定的配体-受体动力学联系起来。相互作用动力学将用一种成熟的方法来监测，即全内反射-荧光光漂白回收。

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.