Collaborative Research: Structural and Molecular Mechanisms for Protein Repair in Photosynthetic Membranes

在我们对光合作用的理解中，一个重要的知识缺口是植物如何修复光系统II (PSII)，即捕获光能的多蛋白全息复合物。该项目的目的是确定PSII磷酸化改变类囊体超微结构的机制，并使受损的PSII能够有效地从密集堆积的颗粒膜转移到非堆叠的类囊体膜，在那里它可以被修复以回收。核心假设是PSII磷酸化引发了间质侧颗粒直径的减少和相邻颗粒膜的分离，从而促进了受损PSII的运动。此外，假设蛋白质磷酸化通过触发大质量全息复合物的拆除和产生静电排斥来增加PSII的迁移率。这项研究的基本原理是，一旦了解了蛋白质磷酸化调节PSII迁移的机制，就可以更好地了解植物如何维持其光合机制以实现最佳生长和发育。通过追求两个具体目标来检验中心假设。目的1：通过低温电子显微镜确定类囊体超微结构磷酸化特异性变化。目的2：确定揭示磷酸化如何在颗粒中动员PSII的分子机制。该方法是在各种修复条件下通过光漂白后荧光恢复（FRAP）测量扩散数据，然后将这些结果与蒙特卡罗模拟开发的FRAP模型进行比较。这些方法将导致以下预期结果。结果1：预期蛋白磷酸化诱导颗粒直径减小，相邻颗粒盘之间的距离变大。这两种超微结构变化都有利于受损PSII向基质层迁移。结果2：预期磷酸化会通过分解和静电斥力加速受损PSII的横向扩散。这些结果有望深入了解PSII降解的机制，以及为什么需要蛋白磷酸化来动员受损的颗粒寄主PSII。本研究对理解许多其他生物膜的分子动力学具有更广泛的影响，因为潜在的机制可能是相似的。该项目的社会效益是，更好地了解植物如何维持其光合作用机制，将有助于提高作物在粮食和生物燃料方面的健壮性和效率。该项目为两名本科生在令人兴奋的光合作用研究领域提供了培训机会。将努力让来自科学领域代表性不足群体的本科生填补这些职位。本项目将利用电脑电影直观的特点，生成一个基于电脑的教学平台。专题课程将被开发为一个新的基于互联网的课程，名为“运动中的分子”。本课程将面向华盛顿州立大学的研究生以及世界各地的其他学生开放

Intellectual Merit An essential gap of knowledge in our understanding of photosynthesis is how are plants able to repair photosystem II (PSII), the multi-protein holocomplex which captures light energy. The objective of this project is to determine the mechanisms by which phosphorylation of PSII alters the thylakoid ultrastructure and enables damaged PSII to be efficiently mobilized from densely packed stacked grana membranes to unstacked thylakoid membranes where it can be repaired for recycling. The central hypothesis is that PSII phosphorylation triggers a reduction in the grana diameter and separation of adjacent grana membranes on the stromal side thereby facilitating the movement of damaged PSII. Furthermore it is hypothesized that protein phosphorylation increases PSII mobility by triggering the dismantling of the massive holocomplex and by generating electrostatic repulsion. The rationale of the proposed research is that, once the mechanisms by which protein phosphorylation regulates PSII mobility are known, a better fundamental understanding is available of how plants maintain their photosynthetic machinery for optimal growth and development. The central hypothesis is tested by pursuing two specific aims. Aim 1: Determine phosphorylation-specific changes in the thylakoid ultrastructure by cryo-electron microscopy. Aim 2: Determine the molecular mechanisms revealing how phosphorylation mobilizes PSII in grana. The approach is to measure diffusion data by fluorescence-recovery after photobleaching (FRAP) under various repair conditions and then to compare these results to a FRAP model developed by Monte Carlo-simulations. These approaches will lead to the following expected outcomes. Outcome 1: It is expect that protein-phosphorylation induces a reduction of the grana diameter and that the distance between adjacent grana discs becomes larger. Both ultrastructural changes would facilitate migration of damaged PSII to reach stroma lamellae. Outcome 2: It is expect that phosphorylation accelerates lateral diffusion of damaged PSII by disassembly and electrostatic repulsion. These results are expected to yield an in-depth understanding on the mechanisms of the PSII degradation and also why protein phosphorylation is required for mobilization of damaged grana-hosted PSII. Broader Impact This research has a broader impact for the understanding of the molecular dynamics in many other biomembranes since the underlying mechanisms are likely to be similar. The societal benefit of the project is that a better understanding of how plants maintain their photosynthetic machinery will help to increase the robustness and efficiency of crop plants for food and biofuel prospects. The project provides training opportunities for two undergraduate students in the exciting field of photosynthesis research. Efforts will be made to fill these positions by undergraduates from groups under-represented in the sciences. The project will take advantage of the strong intuitive character of computer movies to generate a computer-based teaching platform. Special topics courses will be developed as a new internet-based course entitled "Molecules in Motion." This course will be accessible to graduate students of Washington State University as well as to others throughout the world