International Collaboration in Chemistry: Time-resolved Studies of Endothelial Nitric Oxide Synthase Catalytic Mechanism Using Photoactive NADPH Analogues

University Of Texas Health Science Center San Antonio, San Antonio TX

Investigators

Abstract

Dr. Linda Roman at the University of Texas Health Science Center, San Antonio, USA, is supported by the Chemistry of Life Processes Program in the Division of Chemistry, for an International Collaboration in Chemistry (ICC) award that comprises an international collaboration with Dr. Anny Slama-Schwok, Professor Joanne Xie and Dr. Eric Deprez, who will be supported by Agence Nationale de la Recherche in France. With this award, the Chemistry of Life Processes Program is funding Dr. Linda Roman from the University of Texas Health Science Center in San Antonio and Dr. Jung Ja Kim from the Medical College of Wisconsin to develop probes for and undertake an investigation of the mechanism of nitric oxide synthases. The nitric oxide synthases (NOS) generate nitric oxide (NO), which plays key roles in many physiological processes. The endothelial nitric oxide synthase (eNOS) generates NO in the cardiovascular system, where it is a potent vasodilator. Similarly, the neuronal isoform (nNOS) is involved in neurotransmission and skeletal and cardiac muscle function, and the inducible NOS (iNOS) is expressed in response to an immune challenge. The investigators will study the mechanism of function of these enzymes with the use of customized photoactivatable probes. The ability to modulate the activities of these enzymes will help elucidate their physiological roles and regulation, as well as give insight into their mechanisms. This pursuit will allow undergraduate students and postdoctoral fellows to acquire specialized training in protein expression, purification, characterization, and in the use of X-ray diffraction to determine the protein structure. Specific compounds that bind to the NOSs will also be designed to help achieve these goals, exposing trainees to synthetic chemistry techniques. This project focuses on the use of novel photoactivatable NADPH analogues, called nanotriggers (NTs), targeted to the NADPH site of nitric oxide synthase (NOS) isoforms and cytochrome P450 reductase (POR), to elucidate the mechanism of electron transfer through these enzymes as well as stabilize them in a closed conformation, facilitating crystallization. This project will: (1) determine the X-ray structure of various NT-protein complexes; (2) elucidate time-resolved structure/function studies of the first catalytic steps by kinetics studies in solution and time-resolved X-ray crystallography; (3) design novel isoform-specific eNOS activators by in silico simulations; and (4) monitor eNOS trafficking in cells by biphotonic excitation, due to the intrinsic imaging properties and specificity of the NT probes. The photoactive tools to be developed and characterized can trigger a specific catalytic event upon irradiation with a laser pulse. The laser pulse occurs at zero time, allowing for synchronization of initiation of catalysis, which can be monitored in a time-resolved manner. This proposal will develop new tools for time-resolved studies of selected proteins that overcome the limitation of slow (greater than or equal to ms) diffusion of the probe by binding directly to the protein with the ability to trigger catalysis by ultrafast electron injection to the protein. This approach represents a new tool to synchronize an ensemble of enzymes in solution induced by a laser pulse and represents an alternative to single molecule studies.