NSF Award
1708829
With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Dr. Carey Johnson from the University of Kansas, Dr. Brian Smith from Medical College of Wisconsin, and Dr. David Arnett from Northwestern College. The project investigates shapes (conformations) and shape changes of the enzyme nitric oxide synthase (NOS). The importance of conformational changes in the function of complex enzymes like NOS has become increasingly recognized. NOS activity requires a sequence of electron transfers between different domains of the enzyme. The hypothesis is that these events are controlled by conformational dynamics. In this project, the conformations and conformational changes of NOS are tracked by time-resolved and single-molecule fluorescence techniques. Mass spectrometry shows how domain interactions are regulated by binding to NOS of a calcium signaling protein, calmodulin. The results may show how multi-domain enzymes function through conformational changes. The project provides training in advanced fluorescence and mass spectrometric techniques for graduate students at Kansas University and the Medical College of Wisconsin. Undergraduate students from all participating institutions are trained in interdisciplinary science research. The project also offers training for members of the UKanTeach program which trains science majors as high school science teachers.<br/><br/>This project exploits the spatial and temporal resolution inherent in time-resolved and single-molecule fluorescence to identify and track the conformational states of nitric oxide synthase (NOS). NOS is a homodimeric enzyme that catalyzes formation of nitric oxide by shuttling electrons from modules in one monomer to the heme in the oxygenase domain of the other. Electron transfer in both domains depends on calmodulin binding. Because electron transfer only occurs when the electron donor and electron acceptor are in close proximity, conformational changes must occur to bring different donor and acceptor couples together sequentially. The time scales of interchange among conformational states are not known, nor is it clear how the observed conformational changes correlate with the catalytic cycle of the enzyme. The methods of the project correlate conformations and dynamics detected by fluorescence with knowledge of inter-domain interactions at the peptide level. These inter-domain interactions are observed from hydrogen-deuterium exchange (HDX) mass spectrometry. To identify and assign conformational states observed, selected site-directed mutants that disrupt specific subdomain interactions are probed by both fluorescence and mass spectrometry. The project may lead to a detailed model of NOS function that describes the conformational sequences and rates of conformational changes. The project may also demonstrate how the conformational sequences are related to enzyme regulation. The project provides training in advanced fluorescence and mass spectrometric techniques for graduate students at Kansas University and the Medical College of Wisconsin. Undergraduate students from all participating institutions are trained in interdisciplinary science research. The project also offers training for members of the UKanTeach program which trains science majors as high school science teachers.
