Abstract
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.
Highlights
Photosynthesis is the process of converting light energy to chemical energy carried out by all plants and photosynthetic algae
We established a protocol to produce and refold recombinant Physcomitrella patens Photosystem II subunit S (PsbS) in milligram quantities that can be used for thorough structural characterization by NMR spectroscopy or X-ray diffraction, which techniques are highly demanding in terms of required protein quantities
To the best of our knowledge, we are the first to report the association of recombinant PsbS into native-like dimers, and analyzed the oligomeric states and conformational structure of PsbS by a combination of SDS-gel analysis, dynamic light scattering (DLS), size exclusion chromatography (SEC), diffusion-ordered spectroscopy (DOSY) and hetero nuclear single quantum coherence (HSQC) NMR spectroscopy
Summary
Photosynthesis is the process of converting light energy to chemical energy carried out by all plants and photosynthetic algae. One such mechanism allows plants to adapt to surplus light by dissipating the excess light energy as heat[2] This process is termed as non-photochemical quenching (NPQ) which takes place primarily in the antenna associated with photosystem II (PSII) complex located in the thylakoid membrane of chloroplasts[3]. The most important aspect for NPQ is the existence of a proton gradient across the thylakoid membrane, which senses the photosynthetic state during varying light conditions. The discovery of the membrane protein Photosystem II subunit S (PsbS) made it clear that this protein has a central role in sensing the thylakoid luminal pH and activating a series of complex structural rearrangements that lead to chlorophyll (Chl) de-excitation in the antenna, which is the basis of NPQ5,6. Experiments were carried out at neutral and low pH, mimicking the inactive and active state of PsbS, to understand how PsbS folding and assembly is controlled by the pH environment
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