Exploring the conformations of nitric oxide synthase with fluorescence.

Frontiers in Bioscience (Landmark Edition)
David C ArnettCarey K Johnson

Abstract

Multi-domain oxidoreductases are a family of enzymes that catalyze oxidation-reduction reactions through a series of electron transfers. Efficient electron transfer requires a sequence of protein conformations that position electron donor and acceptor domains in close proximity to each other so that electron transfer can occur efficiently. An example is mammalian nitric oxide synthase (NOS), which consists of an N-terminal oxygenase domain containing heme and a C-terminal reductase domain containing NADPH/FAD and FMN subdomains. We describe the use of time-resolved and single-molecule fluorescence to detect and characterize the conformations and conformational dynamics of the neuronal and endothelial isoforms of NOS. Fluorescence signals are provided by a fluorescent dye attached to the Ca2+-signaling protein calmodulin (CaM), which regulates NOS activity. Time-resolved fluorescence decays reveal the presence of at least four underlying conformational states that are differentiated by the extent of fluorescence quenching. Single-molecule fluorescence displays transitions between conformational states on the time scales of milliseconds to seconds. This review describes the type of information available by analysis of time-resolv...Continue Reading

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