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.