Bacterial Phytochromes for Bimodal Control of Cyclic Nucleotide Signaling

光敏色素（Phytochrome，Phy）是一类对红光和远红光产生生物反应的感觉光感受器。光子吸收驱动生物活性不同的Phy Pr和Pfr状态之间的双向光转换。植物Phys主要通过光依赖性蛋白质：蛋白质相互作用发挥下游反应，而细菌光敏色素（BphP）在调节酶活性方面表现出色。BphPs的光敏光敏核心模块（PCM）可以耦合到固有的光惰性酶，赋予它们光敏性。核苷酸环化酶和磷酸二酯酶（PDE）是合成和分解第二信使3 ′，5 ′-环腺苷和鸟苷的酶，因此受到红光和远红光的调控。然而，迄今为止，催化转换和光调节的程度是有限的。基于BphPs的工程和机制的最新进展，该提议因此寻求设计增强的光调节酶，用于精确双峰控制环核苷磷酸盐（cNMP）水平和下游生理过程。基于BphP的cNMP环化酶和PDE通过PCM和效应子的模块化替换、通过连接这些模块的接头的变化以及通过产生杂合PCM来获得。筛选平台有效地识别改进的变体，并允许它们的酶特性和结构合理化。部署在哺乳动物细胞中，候选BphP环化酶和PDE光遗传学调节cNMP水平和离子通道开放。为了前瞻性地实现上级空间分辨率和更好的组织穿透性，我们研究了利用微米辐射通过双光子吸收来驱动BphPs。改进的基于BphP的cNMP环化酶和PDE使光遗传学成为可能，因为它们不需要外源性发色团，它们支持双向切换以增强时间和空间分辨率，并且它们对穿透组织更深的相对较长的波长作出响应。此外，识别基于BphP的致动器的设计规则代表授予对信号转导的一般洞察力，并告知光调节受体的工程。

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.