Annual SUMMER SCHOOL IN MOLECULAR BIOPHYSICS AND SYSTEMS BIOLOGY

Expression and purification of photosynthetic protein PsbR for crystallization The PsbR protein is a nuclear-encoded photosystem (PS) II subunit specifically found in higher plants, in particular this 10 kDa protein is a component of oxygen evolving complex (OEC) of PSII. The role of PsbR has not been clarified yet. It is reported that PsbR might have auxiliary role for the PsbP binding to PSII or cooperate with manganese ions having complex, where the O2 is formed. Also previous studies showed this hydrophobic protein is located in luminal side of the thylakoid membrane but the exact position, region of the contact with OEC and function of the PsbR remains unknown. PsbR diffracting crystals from higher plants were not 2013 Current PhD student of molecular biology and genetics at the University of South Bohemia 2011 2013 Master student of molecular and evolutionary biology in the University of South Bohemia obtained in order to discover their precise 3D structure, for this reason the aim of this work was to obtain and isolate the PsbR protein for crystallization. PsbR has been expressed in Escherichia coli BL21. A plasmid vector pET41a(+) allowed expression of the PsbR as a glutathione-S-transferase (GST) fusion protein, after that it was cleaved from the carrier protein with thrombin (1 NIH/mg protein) and purified on glutathione sepharose high performance column using FPLC AKTApurifier system. We adopted the GST expression system and modified it for production of electrophoretically pure PsbR protein in sufficient quantities (~mg) to be used in crystallography experiments and others structural studies. In this work we performed measurements of circular dichroism (CD) of the PsbR that confirmed that PsbR contains α-helix.