Using 4-Cyanophenylalanine to Probe the Degree of Water Exposure in a Peptide Hydrogel

Abstract

Nitric oxide (NO) is an essential signaling molecule for neurotransmission, cardiovascular function, and cellular defense. However, NO overproduction from neuronal nitric oxide synthase (nNOS) increases infarct size after ischemic stroke and has been linked to the pathogenesis of Alzheimer's and Parkinson's disease. Despite fundamental roles of NO in cellular signaling and disease, intact nNOS holoenzyme structures have remained elusive, and the structural basis for the electron transfer cycle across the reductase and oxygenase domains is unclear. In previous studies, we used negative-stain electron microscopy (EM) to determine conformational states and the overall domain organization of nNOS during its catalytic cycle. In current work, we have interrogated the nNOS and nNOS:Calmodulin (nNOS:CaM) complexes by cryogenic-EM (cryo-EM). In these intact complexes, the oxygenase dimer structure was determined to $4Å , revealing its active-state architecture, while the nNOS reductase domains were identified to be flexibly linked in both CaM-free and CaM-bound states. Additional structural analysis of crosslinked-stabilized complexes, including 2D and 3D classification, will be presented that reveals the overall architecture and highlights the use of cryo-EM methods in elucidating different conformations of the nNOS:CaM complex.