Carey K. Johnson
Professor, Department of Chemistry
University of Kansas

 

 

Understanding conformational control of nitric oxide synthase activity (2017-18)

Nitric oxide synthase (NOS) is a multi-domain, homodimeric enzyme that generates the signaling molecule nitric oxide. NOS is activated by binding of the calcium-signaling protein calmodulin (CaM) to trigger the sequential transfer of electrons between subdomains of the enzyme. Electron transfer is a short-range process, so enzyme function requires formation of a sequence of conformations that place electron-transfer donors and acceptors in close proximity. The project is based on the hypothesis that NOS activity is regulated by conformational dynamics. Experimental methods are needed to probe conformational dynamics, which remain largely uncharacterized. The combination of fluorescence lifetime and singlemolecule fluorescence measurements offers a unique capability to track conformational interchange. A fluorophore will be attached to CaM. Sensitivity to conformation arises from Förster resonance energy transfer (FRET) to the heme group of NOS. The long-range goal of this project is to map out the conformational dynamics, including the sequence of conformational states, in the endothelial and neuronal isoforms of NOS. The Specific Aims for the current project are:

1. Assign conformational states detected in fluorescence to specific subdomain interactions based on site-directed mutations that disrupt specific interaction. Fluorescence lifetime states (detected in previous results) will be assigned to specific inter-domain interactions by comparison of the lifetimes for a sequence of site-directed mutations known to disrupt domain interactions.

2. Carry out control experiments to demonstrate conformational interchange in single-molecule fluorescence time sequences. Single-molecule fluorescence time sequences have been recorded showing interchange among multiple fluorescence states. Experiments are proposed to demonstrate that the observed fluorescence dynamics in fact report protein conformational dynamics.

Successful completion of the project will establish crucial preliminary results that will be important for submission of a successful R01 NIH proposal.

Tracking Nitric Oxide Synthase Conformations and Dynamics   (2015-2016)

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.