Crystallization of photosystem II core complex of Pisum sativum

 

K. Shamayevaa, T. Prudnikovaa, J. A. Gavirac, E. Pineda Molinac, P. Rezacovad, I. Hunalovae, F. Vachaa,e, M. Kutya,b, J. M. Garcia-Ruizc and I. Kuta Smatanovaa,b,*

 

aInstitute of Physical Biology University of South Bohemia Ceske Budejovice, Zamek 136, 373 33 Nove Hrady, Czech Republic

bInstitute of Systems Biology and Ecology, Academy of Science of Czech Republic, 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 Academy of Science of Czech Republic, Flemingovo n. 2, 16637 Prague, Czech Republic

eBiology Centre, Institute of Plant Molecular Biology, Academy of Science of Czech Republic, Branisovska 31, 370 05 Ceske Budejovice, Czech Republic

 

Keywords: biocrystallization, membrane protein, core complex photosystem II

Introduction:

Membrane proteins often play important roles in fundamental processes of life. Various methods such as electron microscopy, biochemical and biophysical spectroscopy methods, or electron diffraction have been used to study membrane proteins, yet to determine their structure, in most of the cases, remains problematic [1].

Photosystem II (PSII) is a multisubunit membrane protein complex performing light-induced electron transfer and water-splitting reactions, leading to the formation of molecular oxygen [2].

The goal of our work is to find suitable crystallization conditions, grow protein crystals and solve a protein structure using X-ray diffraction technique.

 

Material and Methods:

OEC PSII was isolated from green pea (Pisum sativum) and purified according to Ghanotakis et al. [3]. The purification procedure was later improved similarly to Kern et al. [4]. The SDS-PAGE electrophoresis in a buffer system of Laemmli [5] using 12% acrylamide resolution gel was used for analysis of isolated complexes. The protein solution was concentrated to 2–3 mg/ml of chlorophyll a (~1.7–2.7 mM Chla) and supplemented with 1 mM MnCl2 prior to crystallization trails.

 

Results:

Protein has been crystallized using A) vapor diffusion methods as well as B) counter-diffusion [8]. Initial crystallization screening has been performed in sitting drops [6-7] for method A) and in single capillaries [9] for B). Various commercial screens (MembFacTM and ‘‘Crystal ScreenTM of Hampton Research (Aliso Viejo, CA, USA) and JBScreen Crystal Screening Kits of JenaBioscience (Jena, Germany)) were tested in individual crystallization trials. Crystallization experiments are in the progress.

 

Acknowledgements:

This work is supported by the Ministry of Education of the Czech Republic (MSM6007665808 and LC06010) and by the Academy of Sciences of the Czech Republic (AVOZ60870520).

References:

1.       Ivana Kuta Smatanova, Jose A. Gavira, Pavlına Rezacova, Frantisek Vacha, Juan M. Garcıa-Ruiz (2007), New techniques for membrane protein crystallization tested on photosystem II core complex of Pisum sativum, 255-259.

2.       Kamiya N, Shen JR (2003) Crystal structure of oxygen-evolving photosystem II from Thermosynechococcus vulcanus at 3.7 Å resolution. PNAS 100:98–103

3.       Ghanotakis DF, Demetriou DM, Yocum CF (1987) Isolation and characterization of an oxygen-evolving photosystem-II reaction center core preparation and a 28 kDa chl-alpha-binding protein. Biochim Biophys Acta 891:15–21

4.       Kern J, Loll B, Lueneberg C, DiFiore D, Biesiadka J, Irrgang K-D, Zouni A (2005) Purification, characterisation and crystallization of photosystem II from Thermosynechococcus elongates cultivated in a new type of photobioreactor. BBA 1706:147–157

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6.       Bergfors TM (ed) (1999) Protein Crystallization: techniques, strategies, and tips. International University Line, La Jolla

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8.       Garcia-Ruiz JM (2003) Counter-diffusion methods for macromolecular crystallization. Methods in Enzymology, vol 368. Academic Press, pp 130–154

9.       Lopez-Jaramillo FJ, Garcia-Ruiz JM, Gavira JA, Otalora F (2001) Crystallization and cryocrystallography inside X-ray capillaries. J Appl Cryst 34:365–370