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/远红光致变色胆蛋白光感受器，主要充当主转录调节剂，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，光敏色素分子本身的实际工作原理在很大程度上尚不清楚。蓝藻光敏色素 Cph1 显示出与植物光敏色素特别密切的相似性，因此代表了光敏色素分子功能研究的最有用模型。然而，此外，Cph1 是一种光子调节的组氨酸激酶，其功能机制一般与“双组分”感觉激酶有关，包括医学上重要的病原体的感觉激酶。在这个项目中，我们将开发和使用先进的魔角旋转 NMR 方法，不仅可以识别原子邻居，还可以推导出 Cph1 内高精度的原子间距离。作为对从 X 射线晶体学获得的详细但大部分静态结构信息的补充，我们将生成 Cph1 母态功能动力学的详细图片，并在冻结捕获的帮助下，在宽时间尺度上生成光转换中间体。定点突变体还将使我们能够通过实验操纵该分子。我们将重点关注分子内信号传导过程中至关重要的区域，即发色团 D 环附近和 PHY 域的舌区域。我们还建议利用 MAS NMR 中的 31P 来研究催化 ATP 结合结构域及其组氨酸靶标。