Ligand Translational Mobility and Concentration Near Membranes

University Of North Carolina At Chapel Hill, Chapel Hill NC

Investigators

Abstract

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.