Bacterial Phytochromes for Bimodal Control of Cyclic Nucleotide Signaling

光敏色素（Phytochromes, Phy）是一类调节对红光和远红光的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态之间的双向光转换。植物主要通过依赖光的蛋白质：蛋白质相互作用发挥下游反应，而细菌光敏色素（BphP）擅长调节酶活性。BphPs的光敏光感核心模块（PCM）可以与内在光惰性酶偶联以赋予其光敏性。核苷酸环化酶和磷酸二酯酶（PDE）产生和破坏通用的第二信使3 ‘,5 ’环腺苷和鸟苷单磷酸，因此受到红光和远红光的控制。然而，迄今为止，催化转换和光调节的程度有限。基于BphPs的工程和机制的最新进展，本提案因此寻求设计增强的光调节酶，用于精确双峰控制环核苷单磷酸（cNMP）水平和下游生理过程。基于bphp的cNMP环化酶和pde是通过模块化替换PCMs和效应物，通过改变连接这些模块的连接物，以及通过生成杂交PCMs获得的。筛选平台有效地识别改进的变体，并允许他们的酶表征和结构合理化。在哺乳动物细胞中，候选BphP环化酶和PDEs光遗传学调节cNMP水平和离子通道打开。为了实现更高的空间分辨率和更好的组织穿透，我们研究了双光子吸收和微米辐射对BphPs的驱动作用。改进的基于bphp的cNMP环化酶和pde支持光遗传学，因为它们不需要外源发色团，它们支持双向开关以提高时间和空间分辨率，并且它们响应相对较长的波长，穿透组织更深。此外，确定基于bphp的致动器的设计规则有助于深入了解信号转导，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.