STUDIES OF CONFORMATIONAL CHANGES IN SOLUBLE GUANYLATE CYCLASE

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Soluble guanylate cyclase (sGC), a 150 kD heterodimeric protein, is the primary nitric oxide (NO) receptor in the cell. NO binds to heme in the N-terminal domain of the beta subunit, stimulates the production of cGMP and leads to NO-dependent signaling cascades. In vitro studies have shown that a probably allosteric conformational change takes place when ligands bind. In addition to the physiological ligand NO, there are effector ligands, typified by YC-1 that act in concert with carbon monoxide (CO). We request beam time to perform small angle x-ray scattering studies on soluble guanylate cyclase in order to characterize ligand binding associated conformational changes. Several different domains and constructs of both human and Manduca sexta sGC have been expressed by recombinant methods and are available, or will become available in the near future. Preliminary analytical ultracentrifugation studies demonstrate that the conformational change is large enough to be characterized by SAXS and that the protein does not aggregate significantly under the conditions of the centrifugation experiment.