Crystallization study of high plants photosystem II and chlorosomal

bacteriochlorophyll c aggregates

Tatyana Prudnikova^a, José A. Gavira^c, Pavlína Řezáčová^d, Michal Kutý^{a, b}, František Vácha^{a, e}, Jakub Pšenčík^f, Juan M. García-Ruiz^c and Ivana Kutá Smatanová^{a, 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

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

^fCharles University, Faculty of Mathematics and Physics, Ke Karlovu 3, CZ-121 16, Czech Republic

Photosynthesis realized by photosystem II (PS II) uses light energy to couple the formation of molecular oxygen to the fixation of carbon dioxide. It consists of four membrane-internal subunits (D1, D2, CP43, CP47), several smaller internal membrane (including PsbE and PsbF, constituting cyt b-559) and three external subunits (PsbQ, PsbP, PsbO in green algae and higher plants). PS II is located in the thylakoid membrane of higher plants, algae and cyanobacteria.

Chlorosomes are the main light harvesting complexes of green
photosynthetic bacteria. Typical chlorosome is an ellipsoidal body (100-200 nm x 20-50 nm) which consists of bacteriochlorophyll (c, d or e) molecules, carotenoids (chlorobactene), very small amount of quinones (menaquinone-7), lipids (monogalactosyl diglyceride) and proteins. The main difference from other light harvesting complexes is that the main pigments aren’t associated with protein and self-assemble into aggregates.

The aim of our work was based on using advanced counter-diffusion and standard vapor-diffusion methods, to observe capability of individual precipitants to influence the crystals growth.

Using advanced 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 mass, glycerol, MPD) to find suitable conditions to produce single crystals of diffraction quality. Crystals of hexagonal shape and needles obtained from different conditions were measured at the synchrotrons DESY, Hamburg (Germany), EMBL, Grenoble (France) and diffractometer Granada (Spain).

References:

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

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

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.

Figure 1. Crystals of Higher Plants Photosystem II.