Higher plant photosystem II
Olga Shmidta, Jaroslava Kohoutovaa, Estela Pineda Molinac, Jose A. Gavirac, Pavlina Rezacovad, David Kaftana, Michal Kutya, b, Juan Manuel Garcia-Ruizc and Ivana Kuta Smatanovaa, b
aInstitute of Physical Biology, University of South Bohemia in Èeské Budìjovice, Zámek 136, 373 33 Nové Hrady, Czech Republic
bInstitute of Systems Biology and Ecology of the Academy of Science of the Czech Republic, v.v.i., Zámek 136, 373 33 Nové Hrady, Czech Republic
cLaboratorio de Estudios Cristalográficos, Edf. López Neyra, P.T. Ciencias de la Salud, Avenida del Conocimiento s/n, 18100 Armilla (Granada) Spain
dInstitute of Molecular Genetics of the Academy of Science of the Czech Republic, v.v.i., Flemingovo n. 2, 16637 Prague, Czech Republic
The aim of this work carried out in collaboration with Laboratorio de Estudios Cristalográficos, Granada, Spain and Institute of Molecular Genetics of the Academy of Science of the Czech Republic, Prague is to crystallize higher plant PS II for high resolution X-ray diffraction and to resolve the structure of the supercomplex.
A combination of centrifugation (sucrose density) and chromatography (ion exchange, gel filtration) techniques was used for extraction and purification of the PSII complexes from solubilised thylakoid membranes of the Pisum sativum L. chloroplasts. The complexes’ activity was followed throughout the isolation routine with optical spectroscopy and polarographic measurements of oxygen evolution rates.
A new protocol for hydroponic plant growth under controlled conditions has already been established as well as re-designed and implemented for isolation of the PS II enriched thylakoid membranes. Broad screening for most suitable buffers and detergents that sustain maximum PSII activity throughout the isolation routine has been already done. Nowadays the protocol for PSII purification is developing. Upcoming methods and approaches for crystallisation of both membrane and soluble proteins will be tested subsequently.
This work is supported by grants COST Xtall LD11011, NSM6007665808 and LC06010 of the Ministry of Education of Czech Republic, the OptiCryst project of the Sixth Frame Work, UE. This is a product of the "Factoría Española de Cristalización", Consolider-Ingenio 2010 project (MEC).