Mutagenesis and expression of nitric oxid synthase

Nitric oxide (NO) has emerged as a major messenger molecule and is involved a wide variety of biological processes such as regulation of blood pressure or neurotransmission. The physiologic effects of NO are mediated through stimulation of soluble guanylyl cyclase, an enzyme that converts GTP to cyclic GMP. Accumulation of intracellular cGMP results in activation of cGMP-dependent protein kinases, inhibition or stimulation of cAMP- phosphodiesterases, and modulation of ion channels. NO is synthesized from the amino acid L-arginine by a family of enzymes termed NO synthases (NOS). The proposed study will focus on the biochemical and biophysical characterization of these enzymes and should contribute to a better understanding of function and mechanism of NO biosynthesis. In course of this project, various NO isozymes (neuronal NOS, endothelial NOS, inducible NOS) will be overexpressed in baculovirus-infected insect cells and/or bacteria, purified by affinity chromatography, and characterized with respect to enzyme functions and structure. In addition to the wild-type isozymes, mutants and fragments of NOS will be expressed to investigate the specific role of various enzyme domains in NO biosynthesis. These techniques should allow to localize and characterize the binding domain(s) for tetrahydrobiopterin, an essential cofactor of NOS, and to investigate the role of the socalled PDZ-domain of neuronal NOS in membrane targeting. Beside these functional studies, attempts will be made to crystallize NOS fragments for structure analysis.