RUI: Domain Shuttle Mechanisms for Catalysis and Control in Nitric Oxide Synthases

点击翻译按钮获取中文摘要

Nitric Oxide synthases produce NO as an important molecular signal or as a defensive cytoxin in organisms ranging from archaea to mammals. Eukaryotic NOS isoforms are large modular enzymes; NO production by signaling enzymes is controlled by calcium regulation of electron flux from NADPH to the oxygenase active site via the biosensor calmodulin. Controlled production of NO is central to many physiological feedback systems. The FMN binding domain delivers electrons as it shuttles between the NADPH dehydrogenase and oxygenase catalytic centers in the enzyme. Electrons enter from the NADPH binding site and are donated at the oxygenase site. Experiments will study the formation of the state in which electrons enter the oxygenase site. The mechanism of the electron shuttle will be described in detail. The project will concentrate on nNOS, a Ca+2/CaM controlled signal generator, and iNOS, which is Ca+2 insensitive, to describe the shuttle mechanism and its activation by calmodulin in detail. The experimental plan emphasizes techniques accessible to advanced undergraduates. The proposal demonstrates intellectual merit by providing new paradigms for NOS function and control and novel tests of central concepts. Underlying concepts include the tethered shuttle model, design of input and output state constructs, and new kinetics models.Broader Impacts As a RUI project, the research program has an integral training component including undergraduate student participation in preparative and experimental work. At least four undergraduate students each summer will participate, and additional students work on the project during the academic year. They will get advanced training and experience in preparative and experimental biochemistry and molecular genetics. Students will learn how to design a project, how to plan and carry out a line of experimental work, and how to analyze and evaluate data. Computationally advanced students will learn the basics of simulation. Students will all have the opportunity to attend a regional or national scientific conference and present work. Preparation of presentations is important because it provides experience in writing, organization, and production of meaningful figures. As a result, about twenty undergraduates with diverse backgrounds will be introduced to science as a living intellectual endeavor. Some of them will be recruited into science and allied professions as a career, but all of them will be better equipped to make decisions about their own career choices and, as citizens, about issues on local and national scale.