The principal objective of this research program is to determine the structural parameters that define the functions of the isoforms of nitric oxide synthase (NOS) in their various milieus. L-Arginine is the single natural substrate for the NOS enzymes, producing both L-citrulline and NO., which serves as a gaseous messenger in effecting neurotransmission, cytotoxicity, or vasodilatation, among other biological effects. Three genes encode the NOS proteins: neuronal NOS (NOS-1; nNOS), inducible NOS (NOS-2; iNOS), and endothelial NOS (NOS 3; eNOS) and a number of other gene products found in various tissues resulting from alternative RNA splicing. All NOS isoforms require NADPH as a source of reducing equivalents for oxygenation of L-arginine to form NO.. The basic chemical mechanisms of NOS isoforms are similar to those demonstrated for cytochrome P450- mediated reactions but the extent of coupling of electron equivalents to the production of metabolites, overall reaction rates, and regulation of catalytic activity vary significantly among the isoforms. By understanding their individual structural properties, specific chemical interventions can be designed to regulate the activities of each of the isoforms. The overall hypothesis is that, despite requiring the identical complement of prosthetic groups and cofactors (FAD, FMN, Fe-protoporphyrin IX, Zn and tetrahydrobiopterin) to catalyze the same enzymatic reaction, NOS isoforms have evolved different sequences and structural properties to accommodate their distinct functions.
The Specific Aims are: 1) to continue examining structural properties at the atomic level, using crystallographic methods, and to determine other biophysical properties of the NOS holoenzymes and derivative domains, using analytical ultracentrifugation, electron microscopy, and high pressure spectroscopy that determine their unique characteristics; 2) to characterize the protein-protein interactions that regulate NOS activities, using various biophysical methods including co-crystallization and surface plasmon resonance techniques, to measure interactions with bradykinin receptors, caveolins, dynamin, and nostrin or their respective interactive domains; and 3) to identify and quantify the various reduced oxygen species (O2-, H202, OONO-) produced by the NOS isoforms under a variety of conditions, including interactions with protein regulators, such as those in the cellular environment. A combination of site-directed mutants, chimeras, and modular constructs to dissect and characterize these processes has been used successfully in performing such studies in this laboratory.
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