UNIVERSITY OF ARIZONA
The primary goal for this grant is to uncover the structural and functional bases for signaling through S-nitrosation. Perhaps the most important target for these studies is the nitric oxide receptor, soluble guanylate cyclase (sGC), a hetrodimeric protein of 15 KDa. Despite much work, stable expression and structural studies have been difficult to achieve for sGC, and little is known about the mechanism behing sGC function. We have succeeded in establishing sGC from the hawmoth as an excellent model for overcoming limitations, a primary goal of specific aim 2 in the parent grant. We have uncovered functional sGC domains that allow for bacterial expression and have achieved small crystals of one such consturct and promising NMR spectra of another. Although this is a promising start, we are slowed by insufficient hands to optimize our constructs, and the lack of dynamic light scattering (DLS) device to optimize conditions for obtaining monodisperse protein, which is required for successful structural studies. We therefore request funds for a DLS insturment and a postdoctroal associate, with supplies, to systematically explore expression parameters. Our request fits with ARRA goals by: 1. Accelerating the pace of our funded research. 2. Creating a new job for a well-trained scientist. 3. Stimulating the economy through the purchasing of new equipment and reagents. Additionally, the proposed work fits well with the 2-year time frame for stimulus funding. Simply increasing the number of parameters we can explore through the hiring of a skilled researcher, and increasing the efficiency of our search for optimal conditions through the purchasing of a DLS device, will allow us to surpass the primary impediment in nitric oxide signaling studies.