Bacterial Phytochromes for Bimodal Control of Cyclic Nucleotide Signaling

光敏色素(Phy)是一类感光感受器，协调生物对红光和远红光的反应。光子吸收驱动了生物活性不同的Phy、Pr和Pfr态之间的双向光转换。虽然植物的下游反应主要是通过光依赖的蛋白质：蛋白质相互作用，但细菌光敏色素(BphP)在调节酶活性方面表现出色。BphP的光敏感光核心模块(PCM)可以连接到固有的光惰性酶上，从而赋予它们光敏性。因此，合成和破坏通用第二信使3‘，5’-环腺苷和鸟苷一磷酸的核苷环酶和磷酸二酯酶(PDE)受到红光和远红光的控制。然而，到目前为止，催化剂的周转率和光调节的程度都是有限的。因此，在双酚P的工程和机制的最新进展的基础上，该提议寻求设计增强的光调节酶来精确地双峰控制环核苷酸单磷酸(CNMP)水平和下游的生理过程。基于BphP的cNMP环化酶和PDE是通过PCM和效应器的模块化替换、通过改变连接这些模块的连接子以及通过产生杂交PCM来获得的。筛选平台有效地识别改进的突变体，并允许它们的酶特性和结构合理化。候选的BphP环化酶和PDE部署在哺乳动物细胞中，光遗传地调节cNMP水平和离子通道开放。为了实现更高的空间分辨率和更好的组织穿透性，我们研究了微米辐射下双光子吸收对BphPs的驱动作用。改进的基于BphP的cNMP环化酶和PDE增强了光遗传学，因为它们不需要外源发色团，它们支持双向切换以提高时间和空间的分辨率，并且它们对穿透组织更深的相对较长的波长做出反应。此外，确定基于BphP的致动器的设计规则将使人们对信号转导有全面的了解，并为光调节受体的工程提供信息。

Phytochromes (Phy) are a class of sensory photoreceptors orchestrating biological responses to red and far-red light. Photon absorption drives the bidirectional photoconversion between the Phy Pr and Pfr states that differ in biological activity. Whereas plant Phys exert downstream re-sponses primarily through light-dependent protein:protein interactions, bacterial phytochromes (BphP) excel in regulating enzymatic activity. The light-sensitive photosensory core modules (PCM) of BphPs can be coupled to intrinsically light-inert enzymes to bestow light sensitivity on them. Nucleotidyl cyclases and phosphodiesterases (PDE), that make and break the universal second messengers 3’, 5’-cyclic adenosine and guanosine monophosphate, have thus been sub-jected to the control by red and far-red light. To date, the catalytic turnover and the degree of light regulation are however limited. Building on recent advances in the engineering and mechanism of BphPs, this proposal hence seeks to devise enhanced light-regulated enzymes for the precise bimodal control of cyclic nucleoside monophoshate (cNMP) levels and downstream physiological processes. BphP-based cNMP cyclases and PDEs are obtained by modular replacement of PCMs and effectors, by variation of the linker conjoining these modules, and by generating hybrid PCMs. Screening platforms efficiently identify improved variants and allow their enzymatic char-acterization and structural rationalization. Deployed in mammalian cells, candidate BphP cyclases and PDEs optogenetically regulate cNMP levels and ion-channel opening. To prospectively ena-ble superior spatial resolution and better tissue penetration, we investigate the actuation of BphPs by two-photon absorption with micrometer radiation. Improved BphP-based cNMP cyclases and PDEs empower optogenetics, since they do not require exogenous chromophores, they support bidirectional switching for enhanced resolution in time and space, and they respond to compara-tively long wavelengths that penetrate tissue more deeply. Moreover, the identification of rules for the design of BphP-based actuators stands to grant general insight into signal transduction and informs the engineering of light-regulated receptors.