Large-scale in vitro production, refolding and dimerization of PsbS in different microenvironments

Plants adapt to fluctuating light conditions by a process called non-photochemical quenching (NPQ), where membrane protein PsbS plays a crucial role and transforms a change in the pH-gradient across the thylakoid membrane under excess light conditions into a photoprotective state, leading to de-excitation of antenna chlorophylls. The PsbS activation mechanism is elusive and has been proposed to involve a monomerization step and protonation of specific residues. To elucidate its function, it is essential to produce PsbS in large quantities, stabilize PsbS in a membrane-mimicking environment and analyze its pH-dependent conformational structure. We present an approach for large-scale in-vitro production and spectroscopic characterization of PsbS under controlled, non-crystalline conditions. We produced PsbS of the moss Physcomitrella patens in milligram quantities in E. coli, refolded PsbS in several detergent types and analyzed its conformation at neutral and low pH by Dynamic Light Scattering and NMR spectroscopy. Our results reveal that at both pH conditions, PsbS exist as dimers or in apparent monomer-dimer equilibria. Lowering of the pH induces conformational changes, destabilizes the dimer state and shifts the equilibria towards the monomeric form. In vivo, a similar response upon thylakoid lumen acidification may tune PsbS activity in a gradual manner.