Conformational Studies of Soluble Guanylate Cyclase Through Time-Resolved Fluorometry

Abstract

We have used time-resolved fluorometry to determine the decay lifetimes of the four tryptophans in soluble guanylate cyclase (sGC). sGC is a heme sensor protein, which is able to be physiologically activated by nitric oxide (NO). Based on our lab's previous work, the binding of NO induces a conformational change, thus activating sGC. Based on this work, a model has been created using steady-state conditions. We are using the time-resolved data, a more sensitive technique to confirm the steady-state model. Time-resolved spectroscopy allows for individual tryptophan identification, as each one has a unique tau value. The unique tau values will allow for testing with the native protein since the tryptophans can be monitored individually. A temperature dependence study was performed to determine whether or not the tau values are temperature sensitive. It was determined that they are very sensitive to temperature, and in order to observe all four tau values, a temperature no higher than 20°C must be used. The four tryptophan's tau values are 0.005 ns, 0.5 ns, 3 ns, and 7 ns. Upon NO binding, although the longest tau value remains unchanged three of the four tau values increase in time. A change in lifetime can indicate multiple changes. Quenching studies are being used to help determine what is happening to the individual tryptophans upon activation. These studies have determined whether the tryptophans are moving farther from the heme or becoming more buried in the protein, providing more information about the conformational change. After the model has been confirmed using NO, the drug activator YC-1 will also be investigated.