ENGINEERING RED-LIGHT ACTIVATED NUCLEOTIDE CYCLASES

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Engineered photoregulated proteins have the potential to revolutionize biomedical research. In a photoregulated protein, a photon absorbed by a chromophore bound to a photoreceptor protein domain affects activity of an output domain. Visible light is practically harmless to mammalian cells, therefore, it can work as a highly specific, and affordable way to regulate protein activities. The spatiotemporal resolution that can be achieved by using photoregulated proteins is unprecedented as a laser beam can be focused not only on an individual cell but on a particular region of the cell. Engineered photoregulated proteins can be broadly used for activation (or inactivation) of proteins of interest in cell cultures, tissues and animal models. Thus far only blue-light photoreceptors have been used for protein engineering. Because of the short wavelengths of light they have low tissue penetration, which drastically limits their utility in animal models of disease. In contrast, bacteriophytochromes absorb red/far-red light, which has much higher tissue penetration capacity than blue light and is currently used in deep-tissue phototherapies. The objective of this application is to provide the proof of principle that a chromophore-binding module of bacteriophytochromes can be used for engineering of red/ far-red light regulated proteins. The goal of this pilot project is to engineer a red-light activated adenylate cyclase (cAMP synthase). The critical role of cAMP in controlling glucose and lipid metabolism as well as neuronal activity makes photoactivated adenylate cyclase a highly desired tool to study neuronal plasticity, progression of diabetes and obesity.