Phytochrome from photochemistry to signalling: MAS NMR studies of Cph1 structure/function at atomic resolution

植物光敏色素是一种红色/远红光变色的胆蛋白光受体，主要作为主要的转录调节因子，从而在植物整个生命周期中切换依赖光的发育过程，包括发芽和开花。然而，光敏色素分子本身的实际工作原理在很大程度上还不清楚。蓝藻光敏色素Cph1与植物光敏色素有很好的相似性，是研究光敏色素分子功能最有用的模型。此外，Cph1是一种受光子调控的组氨酸激酶，其作用机制与一般的“双组分”感觉蛋白激酶有关，包括那些具有重要医学意义的病原体。在这个项目中，我们将开发和使用先进的魔角旋转核磁共振方法，不仅可以识别原子邻居，还可以在Cph1内推导出高精度的原子间距离。作为对从X射线结晶学获得的详细但基本上静态的结构信息的补充，我们将生成Cph1母态功能动力学的详细图片，并在冷冻捕获的帮助下，在广泛的时间尺度上生成光转化中间体。定点突变也将允许我们在实验中操纵分子。我们将重点关注分子内信号传递过程中的关键区域，即发色团D环的邻近区域和PHY结构域的舌状区。我们还建议利用MAS核磁共振中的31P来研究催化的ATP结合结构域及其组氨酸靶标。

Plant phytochromes are red/far-red photochromic biliprotein photoreceptors that primarily act as master transcriptional regulators, thereby switching light-dependent developmental processes throughout the plant lifecycle including germination and flowering. How the phytochrome molecule itself actually works is, however, largely unclear. The cyanobacterial phytochrome Cph1 shows particularly close similarities to plant phytochromes and thus represents a most useful model for studies of phytochrome molecular function. Additionally, however, Cph1 is a photon-regulated histidine kinase whose functional mechanism pertains to that of “two-component” sensory kinases in general, including those of medically-important pathogens. In this project we will develop and use advanced magic-angle spinning NMR methods not only to identify atomic neighbours but to derive highly-accurate interatomic distances within Cph1. Complementary to the detailed but largely static structural information obtained from X-ray crystallography, we will produce detailed pictures of functional dynamics in the Cph1 parent states and, with the help of freeze-trapping, photoconversion intermediates over wide timescales. Site-directed mutants will also allow us to manipulate the molecule experimentally. We will focus on regions crucial in the intramolecular signalling process, namely the neighbourhood of the chromophore D-ring and the tongue region of the PHY domain. We also propose to exploit 31P in MAS NMR to study the catalytic ATP-binding domain and its histidine target.