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

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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