Structural and functional studies of higher plants photosystem II

 

Tatyana Prudnikovaa, Michal Kutýa,b, José A. Gavirac, Peter Palenčára, František Váchaa, e, Pavlína Řezáčovád, Juan M. García-Ruizc and Ivana Kutá Smatanováa, b

 

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

 bInstitute of Systems Biology and Ecology AS CR Zamek 136, 373 33 Nove Hrady, Czech Republic

cLaboratorio de Estudios Cristalografico, Edf. Lopez Neira, P.T. Ciencias de la Salud, Avenida del Conocimiento, s/n, 18100 Armilla, Granada, Spain

dInstitute of Molecular Genetics AS CR, Flemingovo n. 2, 16637 Prague, Czech Republic, current address:  Dep. Biochemistry, UT Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390-8816

eBiological Centre IPMB AS CR, Branisovska 31, 370 05 Ceske Budejovice, Czech Republic

 

Crystallization of macromolecular complexes such as dimeric core complex of Photosystem II (OEC PSII) from Pisum sativum is influenced by many parameters: purity of sample, homogeneity, capability to form crystals, etc. Tendency to produce suitable crystals for diffraction measurement can be optimized by combination of using different crystallization techniques and other physicochemical parameters (precipitants, additives, pH, etc.) influencing crystallization [1, 2].

Using counter-diffusion method and common vapor diffusion techniques we have tested the influence of several salt additives from Hampton Research screening test (Fe, Ca, Ba, Mg, Ca, Mn, Cd, Cu, Co, Cs, Zn, Y, Ni and Sr), detergents (β-DM, C12E8), buffers with different pH (MES, HEPES, Tris, KH2PO4, pH 6.0-8.0), and cryoprotectants (PEG with several molecular weight, glycerol, MPD) to find suitable conditions to produce single crystals of diffraction quality. Crystals of hexagonal shape and needles obtained from different conditions will be measured at the  synchrotrons DESY, Hamburg (Germany) or ESRF, Grenoble (France). 

For computational part of our work interaction energies were calculated by two ways, the point dipole [4] and also point monopole [5] method. The values of interaction energies between transition monopoles, obtained by the point monopole method are generally more precise. Modification of individual transition energies of pigments by so-called electrochromic shifts caused by reduced pheophytin of the D1 branch (Pheo-D1) gave us realistic light-adapted absorption spectra of PSII RC.

We have obtained the temperature dependence of the light-induced difference spectrum under primary acceptor reduction. Almost identical differences in intensities of 298K and 77K-calculated and 277K and 77K-experimental difference absorption spectra clearly supported earlier assumptions [3]. If the molecule of Pheo-D1  is a part of the multimer interaction, its reduction would lead to a change in the exciton interaction and consequently to a change in the optical absorption spectrum. Since the process of exciton interaction is not dependent on temperature and the Pheo-D1reduction does not cause any change in the low temperature CD spectrum, we suppose that the Pheo-D1 molecule is not coupled in the multimer.

References

[1] K.N. Ferreira, T.M. Iverson, K. Maghlaoui, J. Barber, S. Iwata:  Science, 303 (2004) 1831-1838.

[2] I. Kuta Smatanova, J.A. Gavira, P. Rezacova, F. Vacha, J.M. Garcia-Ruiz: Acta Cryst., A61 (2005) 147.

[3] F. Vácha, J. Pšenčík, M. Kutý, M. Durchan and P. Šiffel: Photosynthesis Research, 84 (2005) 297.

[4] V.I. Prokhorenko, D.B. Steensgaard, A.R. Holzwarth: Biophysical Journal, 85 (2003) 3173-3186.

[5] J. C. Chang: Chem. Phys., 67 (1977) 3901-3909.

Acknowledgements.

This work is supported by grants NSM6007665808 and LC06010 of the Ministry of Education of Czech Republic and Institutional research concept AVOZ60870520 of Academy of Science of Czech Republic.