A laser light source and a data system will be purchased to complete a time-resolved fluorescence spectrometer facility which is now being constructed. This facility will provide the capabilities to make time- resolved fluorescence and phosphorescence measurements on biological macromolecules and to subsequently analyze the resulting data sets by global analysis techniques. Unique to this time-resolved fluorescence facility will be the simultaneous acquisition of data from multiple experiments utilizing a multi-channel detection scheme based upon multi- anode microchannel photomultipliers. In this manner, each particular data set collected on this instrumentation will actually result in 4 to 8 complete data sets being obtained. Thus, very high dimensional data sets can be collected in a short period of time. Complementing the accumulation of these high-dimensional data surfaces, is the availability of global analysis software routines capable of the simultaneous analysis of data surfaces with dimensions > 20. Using this approach, many of the numerical difficulties involved in the recovery of the complex decay kinetics observed from biological systems are obviated. The equipment requested is ideally suited for studies of protein structure-function-dynamics relationships, a central theme of the ongoing research projects in the laboratories of the applicants. The studies to be performed using the requested instrumentation involve a multi-dimensional approach utilizing the combination of time-resolved fluorescence or phosphorescence spectroscopy with: X-ray crystallography, EPR, and NMR measurements. Most of the proteins being studied have been cloned and sequenced enabling site- directed mutants to be expressed thereby assisting in the interpretation of the spectroscopic and crystallographic data. This combination of time- resolved spectroscopy with other complementary information sources provides a powerful approach to understanding molecular mechanisms which are important for relating biological structure to biological function. The requested equipment will have an immediate and significant impact on the research projects outlined in this proposal as well as a longer range impact on other projects in the general area of molecular biophysics at Vanderbilt University.
