Thylakoid membranes capture the solar energy and convert it into chemical energies in the chloroplast of plants, algae, and cyanobacteria. The heterogeneous and dynamic two-dimensional (2D) membrane structures are still rather poorly understood, partially because conventional approaches either investigating native membranes in vivo or biochemically studying individual components have limitation in elucidating the complexity of the thylakoid membrane. We recently developed a methodology to reconstitute the whole thylakoid membrane into a patterned substrate-supported model membrane. A mixture of thylakoid membrane purified from spinach and synthetic phospholipid (DOPC) vesicles spontaneously formed a laterally continuous and fluid 2D membrane in the scaffold of the preformed patterned polymeric bilayer. Photosynthetic functions of the reconstituted membrane were assessed by chlorophyll fluorescence arising from photosystem II (PSII) and the generation of NADPH in the presence of water-soluble ferredoxin and ferredoxin-NADP<sup>+</sup> reductase. Closer investigations using atomic force microscopy (AFM), fluorescence lifetime imaging microscopy (FLIM), and low temperature fluorescence are revealing more detailed structural features of the hybrid thylakoid membrane. Chlorophyll fluorescence had a longer lifetime in the hybrid membrane, indicating that light harvesting complex (LHC) is at least partially dissociated from PSII during the reconstitution process. On the other hand, the fluorescence spectrum from photosystem I (PSI) remained unchanged, suggesting that the integrity of PSI was preserved. Overall, the reconstituted hybrid thylakoids offer an excellent platform to modulate and investigate the molecular components, lateral structures, physicochemical properties, and functions of the thylakoid membrane, giving insight into the 2D organization and molecular machinery of photosynthesis.

Hybrid membrane of reconstituted thylakoid membrane and patterned polymeric lipid bilayer scaffold

ACS Spring 2022

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