Reconstitution of membrane protein PsbH into natural algal lipids

Zbyněk Halbhuber1 and Dalibor Štys1

Photosynthesis Research Center, Institute of Physical Biology, University of South Bohemia, Zamek 136, 373 33 Nové Hrady, Czech Republic

 

Study of membrane proteins in their native environment is restricted from the complexity of native membranes, interference with other membrane constituents and other reactions. To understand organization of the biological membranes and the interaction-taking place between proteins, lipids and variable cofactors, artificial membranes are very useful. The PsbH protein is associated with the reaction centre of PSII in higher plants, algae and cyanobacteria. In our study psbH gene from cyanobacterium Synechocystis sp. PCC 6803 was cloned into a plasmid expression vector, which allowed a synthesis of the PsbH protein as a glutathione‑S transferase (GST) fusion protein in E. coli BL21(DE3) cells. Although the exact role of the protein PsbH is not clear, it seems to be important for the structure and function of photosystem II. These structural and functional role could be closely associated with lipidic environment surrounding the protein. Moreover the protein could bind some cofactors e.g. pigments or in literature mentioned carbon dioxide [1].

Lipids were extracted from Synechocystis sp. PCC 6803 using method of Bligh and Dyer [2]. Extracted lipids were used to prepare liposomes by reversed phase evaporation. The detergent mediated reconstitution was performed according to Lévy et al. [3]. Interaction of lipids and other bound compounds was monitored by measurement of circual dichroism. Interaction of chlorophyls and protein was detected by low temperature fluorescence.

 

[1] Komenda J., Lupínková L. & Kopecký J., Eur. J. Biochem., 269 (2002) 610–619.

[2] Bligh E.G. & Dyer W.J., Can. J. Biochem. Physiol., 37 (1959) 911-917.

[3] Levy D., Bluzat A., Seigneuret M. & Rigaud J.L.,  BBA., 1025 (1990)179–190