Department of Biological and Environmental Sciences, Nanoscience Center, University of Jyväskylä, PO Box 35, FI-40014, Finland.
Phys Chem Chem Phys. 2018 Jul 11;20(27):18216-18225. doi: 10.1039/c8cp01696h.
Phytochrome proteins translate light into biochemical signals in plants, fungi and microorganisms. Light cues are absorbed by a bilin chromophore, leading to an isomerization and a rotation of the D-ring. This relays the signal to the protein matrix. A set of amino acids, which is conserved across the phytochrome superfamily, holds the chromophore in the binding pocket. However, the functional role of many of these amino acids is not yet understood. Here, we investigate the hydrogen bonding network which surrounds the D-ring of the chromophore in the resting (Pr) state. We use UV/vis spectroscopy, infrared absorption spectroscopy and X-ray crystallography to compare the photosensory domains from Deinococcus radiodurans, the phytochrome 1 from Stigmatella aurantiaca, and a D. radiodurans H290T mutant. In the latter two, an otherwise conserved histidine next to the D-ring is replaced by a threonine. Our infrared absorption data indicate that the carbonyl of the D-ring is more strongly coordinated by hydrogen bonds when the histidine is missing. This is in apparent contrast with the crystal structure of the PAS-GAF domain of phytochrome 1 from S. aurantiaca (pdb code 4RPW), which did not resolve any obvious binding partners for the D-ring carbonyl. We present a new crystal structure of the H290T mutant of the PAS-GAF from D. radiodurans phytochrome. The 1.4 Å-resolution structure reveals additional water molecules, which fill the void created by the mutation. Two of the waters are significantly disordered, suggesting that flexibility might be important for the photoconversion. Finally, we report a spectral analysis which quantitatively explains why the histidine-less phytochromes do not reach equal Pfr-type absorption in the photoequilibrium compared to the Deinococcus radiodurans wild-type protein. The study highlights the importance of water molecules and the hydrogen bonding network around the chromophore for controlling the isomerization reaction and spectral properties of phytochromes.
光敏色素蛋白将光转化为植物、真菌和微生物中的生化信号。光信号被双氢卟啉发色团吸收，导致 D 环的异构化和旋转。这将信号传递到蛋白质基质。一组氨基酸在整个光敏色素超家族中保守，将发色团保持在结合口袋中。然而，许多这些氨基酸的功能作用尚未得到理解。在这里，我们研究了发色团 D 环在静止（Pr）状态下周围的氢键网络。我们使用紫外/可见光谱、红外吸收光谱和 X 射线晶体学来比较来自放射性球菌的光敏色素 1、来自橙色硫细菌的光敏色素 1 和放射性球菌 H290T 突变体的光敏色素 1 的光敏结构域。在后两者中，D 环旁边的一个保守的组氨酸被苏氨酸取代。我们的红外吸收数据表明，当组氨酸缺失时，D 环的羰基更强烈地被氢键协调。这与橙色硫细菌的光敏色素 1 的 PAS-GAF 结构域的晶体结构（pdb 代码 4RPW）明显相反，该结构未解析出 D 环羰基的任何明显结合伙伴。我们提出了一种来自放射性球菌的 PAS-GAF 的 H290T 突变体的新晶体结构。分辨率为 1.4 Å 的结构揭示了额外的水分子，这些水分子填补了突变产生的空缺。其中两个水分子明显无序，表明灵活性对于光转化可能很重要。最后，我们报告了一项光谱分析，该分析定量解释了为什么与野生型放射性球菌蛋白相比，没有组氨酸的光敏色素在光平衡中没有达到相等的 Pfr 型吸收。该研究强调了发色团周围水分子和氢键网络对于控制光转化和光敏色素光谱特性的重要性。
