Construction of mutants, protein expression, refolding, and purification
This protocol is extracted from research article:
The molecular pH-response mechanism of the plant light-stress sensor PsbS
Nat Commun, Apr 16, 2021; DOI: 10.1038/s41467-021-22530-4

Constructed plasmids of site-directed mutants of P. patens PsbS were purchased from BaseClear B.V.®. Single mutants were constructed in which E71 was replaced by Q (E71Q) or E176 was replaced by Q (E176Q) that are referred to as M1 and M2, respectively. A double mutant in which both E71 and E176 were mutated to Q (E71Q/E176Q) was constructed and is referred to as M1/M2.The plasmids were transformed, and the mutant proteins were overexpressed in E. coli and purified as has been described for WT PsbS in ref. 21. Briefly, the mutant P. patens PsbS genes were inserted into a pExp5-vector containing an N-terminal His6-tag using Gibson assembly technique and overexpression of the target proteins was carried out in E. coli strain BL21(DE3) pLysS. Cultures were harvested 12–14 h after induction with isopropyl-ß,D-thiogalactopyranoside (ITPG) at cell density of 0.3–0.4 and cell pellets were stored at −80 °C until further use. For the NMR experiments, 13C and 15N uniform labeling of WT PsbS and M1/M2 mutant was carried out by protein overexpression using standard minimal media containing 13C-glucose and 15N-ammonium chloride. For purification, cell pellets were washed in buffer containing Triton X100, yielding white precipitates containing unfolded PsbS as inclusion body pellets. The inclusion body pellets were dissolved and incubated in urea buffer (25 °C, 900 rpm shaking for 30 min) followed by centrifugation at 20,000 x g for 10 min. These steps were repeated twice and followed by washing in urea buffer containing 0.05% lithium dodecyl sulfate (LDS) to separate PsbS as pellet from impurities that were contained in the soluble fraction. Finally, the PsbS pellet was dissolved by adding 0.5% LDS and buffer exchange was performed to remove the high concentration of urea and adjust the pH.

WT and mutant PsbS were refolded in n-Dodecylphosphocholine (FC-12) detergent buffers at pH 5.0 and pH 7.5 conditions, using 100 mM sodium acetate (pH 5.0) or sodium phosphate (pH 7.5) buffers. For the refolding step, unfolded protein (~1 mg/mL) was mixed with an equal volume of refolding buffer and heated to 100 °C for 1 min after which FC-12 was added to the mixture and 200 mM KCl was used to precipitate and remove the LDS21. For the FTIR and 2DIR experiments, the proteins were prepared in deuterated detergent buffer equilibrated at pD 7.5 or 5.0. The pD was set with a standard pH meter using the relation pD = pH + 0.4, with pH the measured value on the pH meter.

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