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

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.

SUBMITTER: Krishnan M

PROVIDER: S-EPMC5680255 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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

Krishnan Maithili M Moolenaar Geri F GF Gupta Karthick Babu Sai Sankar KBSS Goosen Nora N Pandit Anjali A

Scientific reports 20171109 1

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 e ...[more]

PMID: 29123155

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Project description:Protein refolding is an important process to recover active recombinant proteins from inclusion bodies. Refolding by simple dilution, dialysis and on-column refolding methods are the most common techniques reported in the literature. However, the refolding process is time-consuming and laborious due to the variability of the behavior of each protein and requires a great deal of trial-and-error to achieve success. Hence, there is a need for automation to make the whole process as convenient as possible. In this study, we invented an automatic apparatus that integrated three refolding techniques: varying dilution, dialysis and on-column refolding. We demonstrated the effectiveness of this technology by varying the flow rates of the dilution buffer into the denatured protein and testing different refolding methods. We carried out different refolding methods on this apparatus: a combination of dilution and dialysis for human stromal cell-derived factor 1 (SDF-1/CXCL12) and thioredoxin fused-human artemin protein (Trx-ARTN); dilution refolding for thioredoxin fused-human insulin-like growth factor I protein (Trx-IGF1) and enhanced fluorescent protein (EGFP); and on-column refolding for bovine serum albumin (BSA). The protein refolding processes of these five proteins were preliminarily optimized using the slowly descending denaturants (or additives) method. Using this strategy of decreasing denaturants concentration, the efficiency of protein refolding was found to produce higher quantities of native protein. The standard refolding apparatus configuration can support different operations for different applications; it is not limited to simple dilution, dialysis and on-column refolding techniques. Refolding by slowly decreasing denaturants concentration, followed by concentration or purification on-column, may be a useful strategy for rapid and efficient recovery of active proteins from inclusion bodies. An automatic refolding apparatus employing this flexible strategy may provide a powerful tool for preparative scale protein production.

Dataset Information

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

Publications

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

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