Stopped-Flow Cd and Fluorescence Spectrometer

Harris, Michael

Abstract

This proposal requests NCRR funds for the purchase of an Applied Photophysics pi* 180 CDF Spectrometer (APP) to facilitate the kinetic studies of the interactions between biological macromolecules, as well as kinetic and thermodynamic studies of their conformational stability. A major goal of biochemical research in the major users' laboratories is to understand the m9lecular nature of a variety of biochemical interactions including protein-protein interactions, protein-nucleic acid interactions, and the folding of both protein and nucleic acids. Many of these studies include detailed structure-function analyses of specific alterations in the biomacromolecules of interest. These interactions are important in both the fundamental biochemical processes of molecular recognition, as well as in different pathological processes. All of these phenomena share detectable spectroscopic signals that correspond either to complex formation, or to conformational change. These processes routinely occur on time scales of between 10 msec. and ca. l minute. This time regime requires a special sample handling unit, i.e. a stopped-flow, to produce the requisite rapid mixing times. The APP spectrometer permits circular dichroism, UV-Vis, fluorescence intensity and fluorescence anisotropy to be monitored simultaneously by the same instrument. This crucial feature permits the spectroscopic changes to be compared directly, facilitating the correlation of these properties to specific elements of structure. The Case Western Reserve University School of Medicine is committed to providing the necessary infrastructure under the auspices of the Molecular Biology Core Laboratory, and has made provisions for the long-term maintenance of instrument. Importantly, there are no instruments currently on the CWRU campus that provide the same capabilities as the APP spectrometer. This presents an obvious limitation to the research programs of the user group. The requested instrumentation will significantly enhance the scope of the major users' funded research and will provide access to site of the art kinetic measurements to the University community.