Deciphering Photosynthetic Protein Networks: A Crosslinking-MS Strategy for Studying Functional Thylakoid Membranes

Photosynthesis, which sustains life on Earth, depends on highly organized yet adaptable protein assemblies embedded in specialized membranes called thylakoids. Understanding how protein complexes dynamically interact within functional photosynthetic membranes is critical for elucidating cellular energetic metabolism. Here, we present an improved crosslinking mass spectrometry (XL-MS) strategy that captures native protein interactions in functional, photosynthetically active thylakoid membranes from Arabidopsis and Spinach. By monitoring photo-physiology parameters, we demonstrate that electron transport remains physiologically active during the crosslinking process, enabling structural interrogation without disrupting native organization. Mapping crosslinks onto known structures confirms the structural integrity of major complexes and highlights previously uncharacterized assemblies. Our findings pave the way for exploring membrane protein networks in situ and set the stage for integrative structural studies of photosynthetic regulation and adaptation.