Soluble guanylyl cyclase (sGC), the nitric oxide (NO) receptor, is a 150 kDa heterodimeric multi-domain protein that contains heme in the β subunit. Binding of NO to heme leads to rupture of the proximal histidine bond, increased catalytic conversion of GTP to cGMP at a distant guanylyl cyclase catalytic domain, and vasodilation through cGMP signaling. The structure of sGC has not been determined, and little is known about the mechanism by which NO binding to heme leads to increased catalysis. The small molecule YC-1 is known to stimulate sGC activity, but the exact YC-1 binding site and mechanism of action are unknown. Using truncated constructs of Manduca sexta (Ms) sGC lacking the catalytic domain, conformational changes upon YC-1 and NO-binding were characterized using analytical ultracentrifugation and small-angle X-ray scattering. Chemical cross-linking and high-resolution mass spectrometry was used to obtain distance restraints which, when combined with homology models, have provided the first model of sGC domain arrangement and revealed important information about domain-domain interactions. Truncated Ms sGC is highly elongated, contains a coiled-coil in a parallel arrangement, and contains a direct interface between the β H-NOX (Heme Nitric oxide/Oxygen binding domain) and the coiled-coil, and between the β H-NOX and α PAS (Per-arnt-sim) domains. Experiments using analytical ultracentrifugation, fluorescence anisotropy and native mass spectrometry have revealed the YC-1 binding site to be located within the α PAS domain. Additionally, measurement of the kinetics of heme loss and the heme reduction potential were performed to investigate the instability of oxidized sGC heme.

Thesis (Ph. D.)--University of Chicago, Committee on Neurobiology, December 2003. / CD-ROM includes PDF files of the entire dissertation and of the figures alone. Includes bibliographical references. Also available on the Internet.

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Diss. (sammanfattning) Uppsala : Univ., 2004. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Washington, 1997. / Vita. Includes bibliographical references (leaves [92]-96).

Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves [78]-86).

No description available.

Some properties of the adenyl cyclase activity in the maturing testis of Oncorhynchus tshawytscha (chinook salmon) were characterized. The enzymic reaction was linear at 30° and at 15° for at least 60 min. The divalent cation requirement of the salmon testis enzyme was reexamined (33). The optimal concentration of Mg was about 10 mM and of Mn2+ was 5 mM; Mn2+ concentrations above 15 mM caused a marked decrease in enzyme activity. A higher maximal activity was achieved in the presence of Mn2+ than in the presence of Mg2+. Stimulation of the enzyme with the optimal concentration of F-, 12 mM, resulted in a 7-fold increase in the reaction rate over the basal activity. In efforts to solubilize the enzyme, it was found that Lubrol PX and Triton X-100 destroyed enzymic activity but Nonidet P40 and Tween 80 did not. The adenyl cyclase activity in salmon testis homog-enates was stable for at least 6 hours at 0° to 4° but was very unstable at 24°; storage of the homogenate for 24 hours at either 0° to 4° or 24° resulted in a total loss of activity. Differential centrifugation of salmon testis homog-enates which were prepared in isotonic medium revealed tnat all subcellular fractions contained some adenyl cyclase activity. About 55% of the activity sedimented at 600g while only 10% of the activity was recovered in the 105,000g supernatant. The 6300g sediment had a very high specific activity compared with the specific activity of the other fractions. The ATP analogue, adenylyl imidodipho3phate (AMP-PNP), tritium labeled in adenosine, was synthesized from tri-butylammonium imidodiphosphate and adenosine-5’ phosphor-imidazolate. Salmon testis adenyl cyclase catalyzed the conversion of AMP-PNP to cyclic AMP. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Nitric oxide (NO), a reactive diatomic gas and a potent signaling molecule, is required for proper cardiovascular functioning. Soluble guanylate cyclase (sGC), a heterodimeric heme protein, is the key intracellular NO receptor protein which, upon NO binding, undergoes conformational changes leading to catalysis and the cGMP signaling cascade. Several small molecules that allosterically stimulate sGC have been developed for treatment of pulmonary hypertension, but little is known about their binding site or how they stimulate activity. This dissertation describes experiments designed to uncover the molecular basis for signal transduction in sGC by NO and small molecule stimulators. The crystal structure of the α-subunit PAS domain from Manduca sexta (Ms) sGC was solved at 1.8 Å resolution revealing the expected PAS fold but with an additional β strand and a shorter Fα helix. CO binding measurements on different Ms sGC N-terminal constructs and the β₁ (1-380) construct revealed that the α-subunit keeps the β₁ H-NOX domain in an inhibited conformation and this inhibition is relieved by removal of the α-subunit or by addition of stimulatory compounds such as compound YC-1. Linked-equilibria measurements on the N-terminal constructs show that YC-1 binding affinity is increased in the presence of CO. Surface plasmon resonance (SPR) studies on the in-vitro biotinylated constructs showed that YC-1 binds near or directly to the β₁ H-NOX domain. Computational and mutational analysis of the β₁ H-NOX domain revealed a pocket important in allostery and drug action. Finally, we show that the coiled coil domain plays an important role in allosteric regulation of the β₁ H-NOX domain and possibly in signal transduction. Our data are consistent with a model of allosteric activation in which the α-subunit and the coiled coil domains function to keep heme in a low affinity conformation while YC-1 binding to the β₁ H-NOX domain switches heme to a high affinity conformation, and sGC to its high activity form.

No description available.