In this proposal, funds are requested to acquire a sensitive modern X-band CW-ESR spectrometer, which will support biomedical research at Cornell (Ithaca) and Weill Medical College of Cornell University. This will replace a 25 years old ESR spectrometer that can no longer be maintained, and it will provide a large increase in sensitivity as well as other benefits. The research projects requiring this spectrometer will investigate protein-membrane interaction of membrane-modifying and viral fusion proteins, as well as Parkinson's disease implicated proteins, and other studies. The research will greatly benefit from nearly an order of magnitude SNR improvement offered by the requested equipment. The NIH supported research includes: (a). Membrane ordering by fusion peptides and curvature-inducing proteins: We will test the hypothesis that increased bilayer ordering is associated with more robust membrane fusion, by comparing the bilayer ordering effect of more fusogenic mutants of wt20 vs. wt20. We will also investigate changes in the headgroup ordering of negatively charged lipids that are induced by their interactions with the positively charged residues contained in two membrane curvature-inducing proteins: amphiphysin and dynamin. (b) Membrane-bound conformations of human synucleins ?-synuclein (?S) binds to a membrane as an extended helix, as opposed to the U-shape form when bound to spherical micelles. Our hypothesis is ?S can alternate between these two structures. We will apply both pulsed and CW ESR to follow aS conformations as a function of lipid and protein concentrations and composition. (c). There are many uncertainties in the itinerary, mechanisms, and membrane-related consequences of lipoprotein-derived cholesterol trafficking. The ESR spin labeling method will be applied to learn how cellular levels and transport of cholesterol in macrophages affect both early and late events of atherogenesis. (d). The ESR spin labeling method will be applied to gain detailed structural and dynamical information about the membrane domains involved in IgE-receptor mediated cell activation. A large effort has been directed toward understanding how these receptors that have been crosslinked by multivalent ligand (antigen) undergo physical changes and become coupled to intracellular signaling molecules during cell activation. (e). Diphthamide biosynthesis: Recently we presented structural and biochemical evidence that the first step of diphthamide biosynthesis in archaea uses a novel iron-sulfur cluster enzyme, DPH2. Biochemical data suggest that unlike known radical SAM enzymes, DPH2 does not form 5'-deoxyadenosyl radicals. Instead, it breaks the other C-S bond of SAM and transfers the 3-amino-3-carboxylpropyl group to EF-2, possibly via a radical mechanism. We plan to spin-trap and identify the intermediates by the shape of the ESR spectra of their adducts with DMPO and possibly other spin-traps. (f) We are constructing phase diagrams for multi-component lipid mixtures. Some of the phase boundaries, as well as the thermodynamic tielines, are best determined by use of ESR spectroscopy of spin-labeled lipid probes.
