ENGINEERING RED-LIGHT ACTIVATED NUCLEOTIDE CYCLASES

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 工程化的光调节蛋白有可能给生物医学研究带来革命性的变化。在光调节蛋白质中，与光感受器蛋白结构域结合的发色团吸收的光子会影响输出结构域的活性。可见光对哺乳动物细胞几乎是无害的，因此，它可以作为一种高度特异和负担得起的方式来调节蛋白质的活动。通过使用光调节蛋白质可以实现的时空分辨率是史无前例的，因为激光不仅可以聚焦到单个细胞上，而且可以聚焦到细胞的特定区域。工程光调控蛋白可广泛用于细胞培养、组织和动物模型中感兴趣的蛋白质的激活(或失活)。到目前为止，只有蓝光感光器被用于蛋白质工程。由于它们的光波长短，组织穿透率低，这极大地限制了它们在疾病动物模型中的应用。相比之下，细菌藻红素吸收红光/远红光，这比蓝光具有更高的组织穿透能力，目前用于深层组织光疗。本申请的目的是提供原理证明，细菌植物色素的发色团结合模块可用于设计红色/远红光调节蛋白。这个试点项目的目标是设计一种红光激活的腺苷环化酶(cAMP合酶)。CAMP在控制糖脂代谢和神经元活性方面的关键作用使光激活的腺苷环化酶成为研究神经元可塑性、糖尿病和肥胖症进展的理想工具。

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.