Structure Determination of Holoenzyme Neuronal Nitric Oxide Synthase and Calmodulin using Chaperone Assisted Crystallography

Nitric oxide synthase (NOS) is essential to a variety of physiological processes because it converts L-arginine and O₂ to nitric oxide and L-citrulline. Nitric oxide is a powerful second messenger that is implicated in a variety of physiological pathways. Nitric oxide synthase is a dimer composed of an oxygenase domain and a reductase domain. Neuronal nitric oxide Synthase (nNOS) is an isomer of NOS and nNOS is regulated by calmodulin (CaM) binding and therefore the intercellular levels of Ca²⁺. The goal of this project is to crystallize holoenzyme nNOS through chaperone assisted crystallography (CAC).  CAC is the inclusion of high affinity monoclonal antibodies to promote crystal-lattice formation.  The hypothesis is that these chaperones will reduce conformational heterogeneity, resulting in holoenzyme nNOS crystals. We have identified four unique chaperones on the scaffold of humanized-4D5 antigen binding fragments(Fabs) termed: A, E, F and H. Fab A was isolated from screening nNOS alone, Fab F and Fab H were isolated from screening nNOS and CaM, and Fab E was isolated for both nNOS and nNOS + CaM.  Preliminary results show that Fab F increases nitric oxide production by ~37%, suggesting that Fab F might reduce conformational heterogeneity, thereby stabilizing nNOS + CaM. Decoding the protein structure of nNOS allows for a more clear understanding its function and can be used as a gateway for intelligent drug design.