Keywords: Membrane proteins, photosystem II, Pisum sativum, crystallisation trials
Membrane proteins are responsible for enzymatic reactions, which play an important role in all fundamental processes of life. Various methods (such as electron microscopy, biochemical and biophysical spectroscopic methods, electron crystallography etc.) have been used to study proteins, yet to determine their structure, in many cases, remains problematic. The fact that protein crystals are quite unstable, highly temperature- and light –sensitive along with complicated composition of membrane proteins are responsible for difficult crystal growing and solving their structure.
Photosynthesis is a main biological energy source on which life on Earth depends. The process of photosynthesis consists of trapping of light energy by protein complexes owing to which electron transfer and proton pumping lead to a production of ATP and NADPH. Photosystem II (PS II) embedded in photosynthetic (thylakoid) membranes of cyanobacteria and chloroplasts of higher plants and algae represents one of such protein complexes. In structure, PS II is a multi-subunit complex of a molecular weight about 600kDa, containing of large number of cofactors such as molecules of chlorophylls, pheophytins, carothenoids, plastoquinons, iron and manganese which together trap, transfer and modulate a solar energy utilisation to drive reduction reactions and synthesise molecular oxygen from water [1,2].
Materials and methods:
Monomeric photosystem II core complex consisting of reaction centre proteins D1 and D2, the chlorophyll-containing inner-antenna subunits CP43 and CP47, a and b subunits of cytochrome b559, several low molecular weight subunits, pigments (molecules of chlorophyll, molecules of pheophytin, molecule of b-carotene) and cofactors (heme and non-heme binding Fe, Mn cluster) and three extrinsic proteins of oxygen-evolving complex (manganese-stabilizing 33kDa protein, 12kDa protein and cytochrome c550)  was isolated from Pisum sativum, purified and concentrated to 4-6 mg/ml chlorophyll a (~ 20-30 mg/ml protein). The protein solution was prepared containing additive 1mM MnCl2 used in crystallisation trials . Freshly isolated and frozen samples of monomeric photosystem II core complex were crystallised using the counter-diffusion technique implemented in single capillaries  and traditional vapour diffusion method in sitting drops. In both cases the protein solution was gelled with tetramethyl orthosilicate (TMOS) or agarose at different concentration. Gel free experiments were prepared in parallel to test the counter-diffusion technique in a clean environment and those in sitting drop were use as blanks. Commercial “MembFacTM” and “Crystal ScreenTM” crystallisation screening kits of Hampton Research (Laguna Niguel, CA, USA) and “JBScreen Crystal Screening Kits” of JenaBioscience GmbH (Jena, Germany) as well as solutions prepared in-house were used for crystallisation trials performed at 283K and room temperature. Different types of precipitants, inorganic salts, different pH values and variant protein:precipitant concentration ratios were tested experimentally. Optimal values of crystallisation parameters (pH around 7.00 and PEG4K as a precipitant) have been already found.
As a general observation all experiments with frozen samples of monomeric photosystem II core complex produce no crystals, only fresh purified and non-frozen protein was suitable for crystallisation trials. Both, the use of gels and crystallisation in capillaries, were proved as useful crystallisation method.
Possible plate crystals of monomeric photosystem II core complex, were grown in sitting drops from precipitant solution containing 10%PEG4000 and 50mM NaCl in 50mN Bis-Tris pH 7.00 at 283K without presence of gel. Needle-shaped crystals of protein were grown in sitting drops from the same precipitant solution in the presence of 10% TMOS and in capillaries in the presence of 0.25% agarose at room temperature. Green crystals of monomeric photosystem II core complex were measured at X13 beamline of synchrotron DESY (Hamburg) at 100K. Crystals diffracted only to 17Å resolution.
Crystallisation experiments on PSII membrane protein complexes are still in the progress.
This work is supported by the Ministry of Education of the Czech Republic (grant LN00A141), by the Grant Agency of the Czech Republic (grant 206/03/D061) and by the joint C.S.I.C. and AS CR project 01CZ0001, in the frame of the co-operation agreement 2003CZ0013.
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