Carey K. Johnson (2015-2016)
Professor, Department of Chemistry
University of Kansas

Tracking Nitric Oxide Synthase Conformations and Dynamics

In mammals NOS is vital for regulation of blood pressure, neuronal signaling, and resisting infection. Bacterial NOS enables infectious bacteria such as Bacilli to resist oxidative stress and antibiotics. Understanding NOS conformations and interactions and how they differ among mammalian isoforms and in bacteria can therefore help design and screen drug candidates. The long-term objective of the proposed project is to detect and characterize conformations and conformational interchange in nitric oxide synthase (NOS). This application focuses specifically on the interaction between the oxygenase domain and its partner reductase.

 

Specific Aim 1 is to test for docking of the calcium signaling protein calmodulin (CaM) to the oxygenase domain of endothelial NOS (eNOS). In mammalian NOS, including eNOS, oxygenase and reductase domains are contained in one multi-domain polypeptide. Recent time-resolved and single-molecule fluorescence results are presented that show the presence of multiple conformational states for eNOS complexed with CaM. These results led to the hypothesis that a conformation with high fluorescence quenching corresponds to CaM docked to the oxygenase domain of one member of the eNOS homodimer. Such a configuration could then place the FMN module of the reductase domain in position for reduction of the oxygenase heme in the adjacent eNOS protomer, as proposed in several recent structural studies. Specific Aim 1 will test this hypothesis and allow the on and off rates of the docked state to be determined. Results suggest that these rates limit the oxygenase activity of the enzyme. A successful outcome would significantly strengthen the interpretation of fluorescence quenching states of the enzyme as a basis for future proposals.

Specific Aim 2 is to detect interactions of bacterial NOS (bNOS) with reductase partners. In contrast to mammalian NOS, bNOS does not possess a dedicated reductase but rather interacts with reductase partners (e.g. flavodoxins) available in the cell. Research has shown that bNOS in Bacilli allows cells to resist oxidative stress and antibiotics. bNOS is therefore a potential drug target. This part of the project would develop time-resolved and single molecule fluorescence methods to detect interaction of bNOS with a reductase partner. The result would open a new experimental perspective on reductase interactions with bNOS with the potential to significantly advance understanding of bNOS function and mechanisms of inhibition.