PHOTOSYSTEM II REACTION CENTRES: ISOLATION, RECONSTITUTION AND CRYSTALLISATION
Župčanová A. a,b, Vácha F. a,b, Kutá Smatanová I. a
aInstitute of Physical Biology USB CB & Institute of Landscape
Ecology AS CR, Zamek 136, 373 33 Nove Hrady, Czech Republic; bInstitute
of Physical Biology USB CB, Zamek 136, 373 33 Nove Hrady, & Institute of
Plant Molecular Biology AS CR, Branisovska 31, 370 05 Ceske Budejovice, Czech
Republic
The photosystem II is a membrane pigment-protein complex consisting of over 17 subunits. It can be divided in three parts according to its function: 1. the reaction centre (RC); 2. inner and outer light harvesting complexes and, 3. the oxygen-evolving complex. RC is a minimum unit capable of charge separation between the primary electron donor (chlorophyll molecule) and primary electron acceptor (pheophytin), followed by stabilisation of separated charge by transfer of electron to plastoquinone. RC of photosystem (PS) II consists of five protein subunits (D1, D2, a and b subunits of cytochrome b-559 and PsbI). The heterodimer of D1 and D2 binds six chlorophylls, two pheophytins, two b‑carotenes, two plastoquinones denoted QA and QB, respectively, and one iron atom.
The
five-chlorophyll reaction centre of photosystem II (5-Chl RC PSII) was isolated
from green pea (Pisum sativum), purified according to Vacha [1] and reconstituted with purified chlorophyll a as described in [2]. Freshly
isolated and frozen samples of 5-Chl RC PSII concentrated to 15-mg/ml (1.3‑mg/ml chlorophyll
a) were subject of crystallization assays using
the counter-diffusion technique implemented in single capillaries [3] and
traditional sitting drops. Different types of precipitants and detergents and different pH values
were tested experimentally. Optimal values (pH 7.00 and PEG4000 as a
precipitant) have been already found. N-dodecyl-b-D-maltoside (DM) was found as
acceptable detergent. Possible green crystals were
tested at synchrotron in Grenoble (beamline ID14-1) at 100K. Crystallisation experiments on PSII
membrane protein complexes are still in the progress.
Acknowledgements:
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.
[1] Vacha,
F., Joseph, D. M., Durrant, J. R., Telfer, A., Klug, D. R., Porter, G. and
Barber, J.: Proct. Natl. Acad. Sci. USA, 1995, 92, 2929-2933.
[2] Zehetner, A., Scheer, H., Siffel, P.
and Vacha, F.: Biochim.
Biophys. Acta 1556 (2002) 21-28
[3]
Garcia-Ruiz, J.M., Gonzales-Ramirez, L.A., Gavira, J.A. and Otalora, F.: Acta
Cryst, 2002, D58, 1638-1642.