Uncovering Signal Transduction in Blood Pressure Regulation by Nitric Oxide

Abstract

Nitric Oxide (NO) is an important signaling molecule in blood pressure regulation. The NO receptor, soluble guanylate cyclase (sGC), produces the secondary messenger cGMP in response to NO binding. Because of its role in blood pressure regulation, sGC is increasingly targeted for drug discovery and several new drugs treating hypertensive individuals are sGC stimulators. Unfortunately, stimulator development is limited by our understanding of NO and simulator induced conformational changes. In the present study, we used lanthanide resonance energy transfer (LRET) to measure distances between sGC domains in truncated M. sexta sGC constructs and assessed the magnitude of distance changes induced by ligand binding. Our strategy was to place a lanthanide binding domain at various locations in the protein and measure changes in lanthanide luminescence in the presence of the quenching domain, His6 with coordinated copper or nickel ion, which quenches luminescence in a distance dependent manner. Terbium luminescence decayed bi-exponentially in the absence of quencher, and displayed a slow phase with a rate constant of ~2.3 ms. Quencher presence altered the time constant for the slow phase, but not the fast phase. Distances obtained from fitting these data indicate sGC is a compact molecule with the coiled-coil ~23 Å from the a H-NOX N-terminal end. However, we must further develop our system to ensure the decays measured reflect the distance between donor and acceptor. In the future, distances will be measured after ligand or stimulators are bound to determine the change in distance, which will give insight into the conformational dynamics of sGC.