Our goal is to expand the capabilities of our lifetime- resolved multifrequency phase fluorometer in order to be able to resolve the conformational heterogeneity and dynamics of biomolecules (i.e., both lipids and proteins). We require i) a laser system capable of providing an intrinsically modulated coherent light source for excitation of a range of different chromophores and ii) a microchannel plate photomultiplier tube (MCP-PMT) for phase sensitive detection at the ultrahigh frequencies characteristic of subnanosecond lifetime comoponents. This instrumentation will enable us to dramatically increase the sensitivity (i.e., ~ 1000 fold) and dynamic range of our instrument, and will provide the resolution necessary to accurately measure the time-dependent terms relating to the fluorescence intensity decay of site-directed fluorophores, allowing us to directly measure the conformational hetergeneity an dynamics of macromolecules. In addition, the higher incident power will allow us to utilize small amounts of material (i.e., < 10 pmoles). This latter capability will permit the initiation of experiments aimed at identifying key regulatory features in highly purified, native membranes (e.g., brain synaptic membranes) at low, nonperturbing concentrations of fluorophores; as well as structure- function relationships on site-directed mutants of the ATP Synthase (CF1CF0), Ca-ATPase, and the Glutamate Receptor.
