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

光敏色素从光化学到信号传导：原子分辨率下 Cph1 结构/功能的 MAS NMR 研究

• 批准号: 417685888
• 负责人: Professor Dr. Jonathan Hughes, Ph.D.
• 金额: --
• 依托单位: Institut für Analytische Chemie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/417685888?language=en
• 关键词: Phytochrome; photochemistry; signalling; MAS; NMR

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.

植物光敏色素是红色/远红色光致变色胆蛋白光受体，其主要充当主转录调节因子，从而在整个植物生命周期（包括发芽和开花）中切换光依赖性发育过程。然而，光敏色素分子本身实际上是如何工作的，在很大程度上还不清楚。蓝藻光敏色素Cph 1与植物光敏色素具有非常相似的结构，是研究光敏色素分子功能的一个非常有用的模型。此外，然而，Cph 1是一种光子调节的组氨酸激酶，其功能机制一般属于“双组分”感觉激酶，包括医学上重要的病原体。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，不仅要识别原子邻居，但在Cph 1推导出高精度的原子间距离。补充详细的，但很大程度上是静态的结构信息，从X射线晶体学，我们将产生详细的图片的功能动力学的Cph 1母态，并在冷冻捕获的帮助下，在宽的时间尺度上的光转换中间体。定点突变也将使我们能够在实验上操纵分子。我们将集中在分子内信号传导过程中至关重要的区域，即发色团D-环和PHY结构域的舌区附近。我们还建议利用MAS NMR中的31 P来研究催化ATP结合结构域及其组氨酸靶点。