Nanoscience Center, Department of Biological and Environmental Science , University of Jyväskylä , Jyväskylä 40014 , Finland.
Department of Chemistry and Molecular Biology , University of Gothenburg , Gothenburg 40530 , Sweden.
J Am Chem Soc. 2018 Oct 3;140(39):12396-12404. doi: 10.1021/jacs.8b04659. Epub 2018 Sep 18.
Phytochrome proteins regulate many photoresponses of plants and microorganisms. Light absorption causes isomerization of the biliverdin chromophore, which triggers a series of structural changes to activate the signaling domains of the protein. However, the structural changes are elusive, and therefore the molecular mechanism of signal transduction remains poorly understood. Here, we apply two-color step-scan infrared spectroscopy to the bacteriophytochrome from Deinococcus radiodurans. We show by recordings in HO and DO that the hydrogen bonds to the biliverdin D-ring carbonyl become disordered in the first intermediate (Lumi-R) forming a dynamic microenvironment, then completely detach in the second intermediate (Meta-R), and finally reform in the signaling state (Pfr). The spectra reveal via isotope labeling that the refolding of the conserved "PHY-tongue" region occurs with the last transition between Meta-R and Pfr. Additional changes in the protein backbone are detected already within microseconds in Lumi-R. Aided by molecular dynamics simulations, we find that a strictly conserved salt bridge between an arginine of the PHY tongue and an aspartate of the chromophore binding domains is broken in Lumi-R and the arginine is recruited to the D-ring C═O. This rationalizes how isomerization of the chromophore is linked to the global structural rearrangement in the sensory receptor. Our findings advance the structural understanding of phytochrome photoactivation.
植物色素蛋白调节植物和微生物的许多光反应。光吸收导致胆绿素发色团的异构化，这触发了一系列结构变化，激活蛋白质的信号结构域。然而，结构变化难以捉摸，因此信号转导的分子机制仍知之甚少。在这里，我们将双色分步扫描红外光谱应用于来自耐辐射球菌的细菌色素蛋白。我们通过在 HO 和 DO 中的记录表明，与胆绿素 D-环羰基的氢键在第一个中间体（Lumi-R）中变得无序，形成动态微环境，然后在第二个中间体（Meta-R）中完全分离，最后在信号状态（Pfr）中重新形成。通过同位素标记的光谱表明，在 Meta-R 和 Pfr 之间的最后一次转变过程中，保守的“PHY-tongue”区域发生了重折叠。在 Lumi-R 中已经在微秒内检测到蛋白质骨架的其他变化。借助分子动力学模拟，我们发现 PHY-tongue 中的精氨酸和发色团结合域的天冬氨酸之间的严格保守盐桥在 Lumi-R 中被打破，精氨酸被募集到 D-环 C═O。这解释了发色团的异构化如何与感觉受体中的全局结构重排相关联。我们的发现推进了对植物色素光激活的结构理解。
