The importance is in details - isolation and crystallization of PSII from higher plants with different contains of detergent
Tatsiana Holubeva1,2, Estela Pineda
Molina3, Jose A. Gavira3, Daryna
Kulik1,2,
Jiří Heler1,2, Jaroslava Kohoutová1,2 and Ivana Kutá Smatanová 1,2,4
1University of South Bohemia in České Budějovice, School of complex
systems FFPW , Zámek 136,
373 33 Nové Hrady
2University of South Bohemia in České Budějovice, Faculty of
Science, Branišovská 31, 37005 České
Budějovice
3Laboratorio de Estudios Cristalográficos, Edf.
López Neyra, P.T. Ciencias de
4Academy of Sciences of the Czech
Republic, Inst. of Nanobiology and Structural Biology
GCRC, Zámek 136, 373 33 Nové
Hrady
holubeva@nh.cas.cz
Photosystem II (PSII) is a multisubunit pigment-protein complex that catalyses
electron transfer from water to the plastoquinone
pool with concomitant evolution of oxygen. PSII consists of around 25 different
types of protein subunits which are organized into two structurally distinct
parts: core complex (D1,D2, CP47,CP43, intrinsic and
extrinsic proteins, small proteins of
unknown function) and peripheral antenna (light-harvesting complex II (LHCII)
proteins).
For isolation of
PSII from higher plants were selected model organisms, such as tobacco,
spinach, peas, haricot and soy. In our experiments we tried two
different plants – spinach Spinacia oleracea and haricot Phaseolus vulgaris.
Growing hydroponic plant under controlled conditions and optimization of
reproducible purification protocol of homogeneous sample suitable for
crystallization is the main aim of our project. We changed purification
protocol and used new methodology without using of detergent TRITON, which
could be problematic for PSII complex crystallization. We got preliminary
results – microcrystals,
which require the optimization of conditions, using different crystallization technics. It was
also shown that the type of detergent and conditions of solubilization
of thylakoids membranes are critical steps for
crystallization, which should be carefully chosen.
This research was supported by the
ME CR (COST LD11011, CZ.1.05/2.1.00/01.0024), by the AS CR and GAJU 141/2013/P.