We propose to acquire a state-of-the-art pulsed-EPR spectrometer at Case Western Reserve University to carry out structural dynamic studies of bio-macromolecular assemblies that include soluble proteins, membrane proteins, and protein-nucleic acid complexes. EPR spectroscopic applications span a wide range of areas, from nucleic acids and biomembranes, to protein research. This technology provides valuable information about biological processes critical for identification of therapeutic targets in human diseases. This approach is used to characterize protein-protein, protein-lipid, and protein-ligand interactions either by using naturally occurring biological metal ion centers or by incorporating site-directed spin-labeling. In the last decade, pulsed-EPR methods have emerged as an exceptionally sensitive and versatile for quantifying conformational changes in large protein complexes in their native environment. The macromolecule dynamics data obtained from pulsed- EPR are complementary to the high-resolution, yet static, information gained from X-ray crystallography and cryo-electron microscopy. Additionally, this approach is ideally suited for systems too large for NMR measurements. The latest developments in the hardware and pulse-protocols have led to remarkable increases in sensitivity and thereby have significantly extended the range of measurable distances and improved the versatility of this method. The Bruker ELEXSYS E580 Q-band spectrometer is capable of CW- and a variety of pulsed- EPR methods, including double electron-electron resonance (DEER) and electron-nuclear double resonance (ENDOR) spectroscopies. DEER is sensitive to spin-spin distance from 15 to ~80 , which is suitable for studying conformational changes in large proteins and nucleic acid complexes. The advanced features of the proposed instrument, which are critical to the success of the proposed experiments, include a) significantly improved sensitivity relative to X-band, which would allow measurement at lower sample concentrations; b) enhanced sample throughput to cater to the growing needs of the structural biologists; c) increased resolution for extending measurable range to much longer distances, which is particularly important for determining global conformational changes. The NIH-funded research projects that will directly benefit from this instrumentation include the structural and dynamic studies of a number of clinically relevant soluble and membrane proteins (ligand- and voltage-gated channels, EGF and insulin receptors, prions, dynamin related proteins, HIV proteins, and immune receptors). The requested equipment will be the first pulsed-EPR spectrometer in the Northeast Ohio area. This technology will provide a state-of-the-art structural biology tool to several NIH-funded investigators to work on fundamental biological problems, expanding the scope of biomedical research and strengthening the multidisciplinary collaborative interactions within our scientific community here in the Greater Cleveland area.
The goal of this proposal is to establish, for the first time, pulsed-EPR capability in the Greater Cleveland area for the determination of biomolecular structures and dynamics. This instrumentation will directly support the needs of several NIH-funded projects on clinically relevant systems of fundamental importance to public health.
