Nitric oxide (NO) signaling pathways mediate diverse physiological functions, including vasodilation, neurotransmission, myocardial function, and platelet aggregation. Soluble guanylate cyclase (sGC) is the primary receptor of NO. Dysfunctions in the NO-sGC signaling pathway can lead to heart disease, erectile dysfunction, stroke, and hypertension. Understanding the molecular details of NO-induced sGC activation is crucial for developing treatments for these disease states. Mammalian sGC is a multi-domain, heterodimeric hemoprotein. Because full-length sGC has proven intractable to high resolution structure analysis (e.g., X-ray crystallography), the domain architecture and NO-induced conformational changes are poorly understood. The specific aims of the research proposed herein are focused on illuminating the domain organization and conformational changes of sGC through parallel, complementary protein mapping approaches. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a powerful strategy for mapping the solvent exposure of protein surfaces by measuring changes in the rates of amide proton exchange with a deuterated solvent. HDX-MS will be used to map the interaction surfaces of sGC domain truncations. Alanine scanning will be performed to define individual residues that are crucial to sGC inter-domain interactions. To characterize the relative orientations of the sGC domains, hydroxyl radical footprinting will be employed. Hydroxyl radicals generated at a reagent tethered near an inter-domain interaction can cleave polypeptide backbones at proximal residues. These cleavage patterns report on the proximities between surfaces of sGC domains. The results of the HDX-MS, alanine scanning, and hydroxyl radical footprinting will be integrated to develop a model of the domain architecture of full-length sGC. Changes in the sGC domain architecture induced by NO stimulus can then be assessed in full-length sGC. HDX-MS will be used to map NO-induced changes in sGC surface accessibility to develop a model of the conformational changes that control sGC cyclase activity.