Phytochromes are biological red/far-red light sensors found in many organisms. Prototypical phytochromes, including Cph1 from the cyanobacterium Synechocystis 6803, act as photochemical switches that interconvert between stable red (Pr)- and metastable far-red (Pfr)-absorbing states induced by photoisomerization of the bilin chromophore. The connection between photoconversion and the cellular output signal involves light-mediated global structural changes in the interaction between the photosensory module (PAS-GAF-PHY) and the C-terminal transmitter (output) module, usually a histidine kinase, as in the case of Cph1. The chromophore deprotonates transiently during the Pr → Pfr photoconversion in association with extensive global structural changes required for signal transmission. Here, we performed equilibrium studies in the Pr state, involving pH titration of the linear tetrapyrrole chromophore in different Cph1 constructs, and measurement of pH-dependent structural changes at various positions in the protein using picosecond time-resolved fluorescence anisotropy. The fluorescent reporter group was attached at positions 371 (PHY domain), 305 (GAF domain), and 120 (PAS domain), as well as at sites in the PAS-GAF bidomain. We show direct correlation of chromophore deprotonation with pH-dependent conformational changes in the various domains. Our results suggest that chromophore deprotonation is closely associated with a higher protein mobility (conformational space) both in proximal and in distal protein sites, implying a causal relationship that might be important for the global large protein arrangements and thus intramolecular signal transduction.

中文翻译：

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发色团的瞬态去质子化影响蛋白质动力学近端和远端到植物色素Cph1中的线性四吡咯发色团。

植物色素是在许多生物中发现的生物红/远红光传感器。原型植物色素，包括来自蓝藻集胞藻6803的Cph1，充当光化学开关，可在由Bilin发色团的光异构化诱导的稳定的红色（Pr）吸收和亚稳态的远红色（Pfr）吸收状态之间相互转换。光转换和细胞输出信号之间的连接涉及光感测模块（PAS-GAF-PHY）与C端发射器（输出）模块（通常是组氨酸激酶）之间的相互作用中的光介导的整体结构变化，的Cph1。发色团在Pr→Pfr光转换过程中会短暂去质子，并伴随着信号传输所需的广泛的整体结构变化。在这里，我们在Pr状态下进行了平衡研究，涉及在不同Cph1构建体中线性四吡咯发色团的pH滴定，以及使用皮秒时间分辨荧光各向异性测量蛋白质各个位置的pH依赖性结构变化。荧光报告基团附着在位置371（PHY域），305（GAF域）和120（PAS域）以及PAS-GAF双域中的位点。我们显示了在不同域中发色团去质子化与pH依赖的构象变化的直接关系。我们的结果表明，发色团去质子化与近端和远端蛋白位点中较高的蛋白迁移率（构象空间）密切相关，这暗示因果关系可能对全局大蛋白排列以及分子内信号转导很重要。