Keywords: Membrane proteins, photosystem II, Pisum sativum, crystallisation trials
Introduction:
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) [3] 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 [4]. Freshly isolated and frozen samples of monomeric photosystem II core complex were crystallised
using the counter-diffusion technique implemented in single capillaries [5] 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.
Results:
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.
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.
References:
[1] Rhe, K. H., Morris, E. P., Barber, J. and
Kühlbrandt, W.: Nature, 1998, 396, 283-286.
[2] Barber, J. and Kühlbrandt, W.: Curr. Opinion in Struct. Biol., 1999, 9, 469-475.
[3] Zouni, A., Witt, H.-T., Kern, J., Fromme, P.,
Krauss, N., Saenger, W. and Orth, P.: Nature,
2001, 409, 739-743.
[4] Adir, N.: Acta
Cryst., 1999, D55, 891-894.
[5] Garcia-Ruiz, J.M., Gonzales-Ramirez, L.A., Gavira, J.A. and Otalora, F.: Acta Cryst, 2002, D58, 1638-1642.