Thesis defended on December 3, 2025 to obtain the degree of Doctor of the Université Grenoble Alpes
Abstract
Photosynthetic organisms constantly adapt to dynamic light environments to optimize energy capture while preventing photo-oxidative damage. However, structural studies of protein complexes involved in these adaptations remain challenging due to the transient nature of protein interactions and the difficulty of preserving physiological conditions
during the analysis process. The current thesis presents an integrative structural biology strategy that combines crosslinking mass spectrometry (XL-MS) with the functional analysis of native membranes. This approach aims to elucidate the dynamic organization and interactome of the Photosystem II (PSII), a membrane-embedded complex central to the photosynthetic processes occurring in the thylakoid membranes of photosynthetic organisms. The first chapter focuses on developing and optimizing experimental tools to enhance the efficiency and reproducibility of crosslinking reactions in isolated and physiologically active thylakoid membranes, where PSII is localized. Trimethylphenylammonium chloride (TMPAC), a cationic amphiphile, was introduced to neutralize electrostatic repulsion between the negatively charged PhoX crosslinker and the thylakoid membrane. TMPAC improved the yield and reproducibility of XL-generated peptides in thylakoid membranes from both plants Arabidopsis thaliana and Spinacia oleracea, without impairing photosynthetic performance as confirmed by photo- physiological measurements based on chlorophyll fluorescence emission.
In chapter two, this enhanced XL-MS platform was applied to analyze the dynamics of thylakoid protein-protein interactions during non-photochemical quenching (NPQ), a key photoprotective process. By inducing NPQ under controlled light/dark cycles, XL-MS analyses mapped the dynamic associations of different complexes (photosystem II, photosystem I, chlorophyll binding antennas, PSBS) with other thylakoid components. The change of specific crosslinks abundance during NPQ induction and relaxation correlated with fluorescence measurements, supporting the role of previous findings in literature related to antenna modulation and energy dissipation. These findings offer a novel framework for reconstructing the molecular timeline of non-photochemical quenching evolution. They reveal the structural reorganization of Photosystem II (PSII) under excess light conditions and confirm the direct involvement of previously hypothesized subunits and new interactors.
The present work pioneers a near-native and quantitative pipeline for studying dynamic membrane-bound protein interactions in photosynthesis. Beyond plant biology, the methodology opens new avenues for system-wide interactome mapping in other organelles, such as mitochondria, under environmental perturbations.