Crystallization of photosynthetic proteins from Pisum sativum – a new crystallization strategy

Ivana Kuta Smatanova^{1, 2}, Jose A. Gavira³ , Pavlina Rezacova⁴, Frantisek Vacha^{1, 5}, and Juan M. Garcia-Ruiz³

¹Institute of Physical Biology USB CB, Zamek 136, 373 33 Nove Hrady, Czech Republic

²Institute of Landscape Ecology AS CR Zamek 136, 373 33 Nove Hrady, Czech Republic

³Laboratorio de Estudios Cristalografico, Edificio BIC-Granada, Avda. de la Innovacion 1, P.T. Ciencias de la Salud, 18100-Armilla, Granada, Spain

⁴Institute of Molecular Genetics AS CR, Flemingovo nam. 2, 166 37 Praha 6, Czech Republic

⁵Institute of Plant Molecular Biology AS CR, Branisovska 31, 370 05 Ceske Budejovice, Czech Republic

The photosystem II (PSII) is a multisubunit membrane-protein complex consisting of membrane in-lying subunits, hydrophilic peripheral subunits and large number of cofactors, including chlorophylls, pheophytins, carothenoids, plastoquinones, iron and manganese, which together trap, transfer and modulate a solar energy to drive catalytic photoinduced oxidation of water and synthesize molecular oxygen. Catalytic mechanism of PSII has been studied using a wide range of approaches [1-3], but particular molecular details of water oxidation catalyzed by the oxygen evolving center (OEC) remains unclear. Crystallographic studies of OEC PSII from thermophilic cyanobacterium [4-6] have given the first description of the structure of PSII, but these models are not absolutely complete as yet.

Finding suitable crystallization condition is the main problem to solve a protein structure by X-ray diffraction techniques. The current crystallization strategies are mainly based on the screening upon previously successful chemical cocktails and on classical crystallization techniques based on evaporation. That strategy has been found partially successful for soluble globular proteins. However, membrane protein is a particular case for which the success rate applying commercial crystallization screens is much lower than for soluble proteins. The fact that membrane proteins are often unstable, highly temperature and light sensitive together with their complicated composition are responsible for difficult crystal growing and solving their structure.

Here we report a new approach for crystallization of monomeric photosystem II core complex (OEC PSII) from green pea. The core complex of PSII was isolated from Pisum sativum, purified and concentrated to 2-3 mg/ml of chlorophyll a (~ 10-15 mg/ml of protein). The protein solution was prepared containing additives 10mM NaCl and 1mM MnCl₂ used in crystallization trials [7]. Freshly isolated and frozen samples of PS II protein complex were crystallized using the counter-diffusion technique implemented in single capillaries [8] and traditional vapor diffusion method in sitting drops. After performing of several series of crystallization experiments it was found that only fresh purified and non-frozen protein is suitable for crystallization trials. Crystallization in capillaries was ascertained as an efficient method to find and optimize crystallization conditions.

The core complex of PSII crystallized in green needle-shaped crystal form from precipitant solution containing PEG4000 and MPD in MES pH 6.50 at 291-293K. Protein character of PSII crystals was confirmed by laser spectroscopy under use of Olympus IX70 inverted microscope (Olympus, Japan) equipped with Triax320 monochromator and CCD camera (Jobin Yvon, France), and by X-ray diffraction measurements. Frozen monocrystals of PSII core complex were tested at the synchrotrons DESY, beamline X13 (Hamburg, Germany) and ESRF, beamlines BM16 and ID14-1 (Grenoble, France) and at the home source diffractometer at IMG AS CR (Prague, CZ). Diffractions at low resolution have also certified protein character of crystals. As the crystals were very small and insufficiently ordered for high resolution analysis, further experiments will be aimed at obtaining better-quality crystals from which the structure of PSII complex from higher plant could be solve.

Acknowledgements:

This work is supported by the Grant Agency of the Czech Republic (grant 206/03/D061), by the joint C.S.I.C. and AS CR project 2004CZ0003 in the frame of the cooperation agreement P2004CZ01, by the Ministry of Education of the Czech Republic (MSM6007665808) and by the Academy of Sciences of the Czech Republic (Institutional research concept AVOZ60870520).

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