Structural analysis of the extrinsic PsbP protein of PSII from Spinacea oleracea by X-ray crystallography, Raman spectroscopy and Bioinformatics

 

Jaroslava Kohoutováa, Vladimıír Kopecký Jr. b, Mikalai Lapkouskia,c, Kateøina Hofbauerováb,d,  Žofie Sovováa, Sergio González-Pérezce,  Ivana Kutá Smatanováa,c, José L. Revueltaf,  Juan B. Arellanoe, Rudiger Ettricha,c

 

aInstitute of Systems Biology and Ecology, Academy  of Sciences of the Czech Republic,  Zámek 136, 37333 Nové Hrady, Czech Republic

bInstitute of Physics, Faculty of Mathematics and Physics, Charles University in Prague, Ke Karlovu 5, Prague 2, CZ-12116, Czech Republic

cInstitute of Physical Biology, University of South Bohemia, Zámek 136, 37333 Nové Hrady, Czech Republic

dInstitute of Microbiology, Academy of Sciences of the Czech Republic, Vídeòská 1083, CZ-142 20 Prague 4, Czech Republic

eDepartamento de Estrés Abiótico, Instituto de Recursos Naturales y

Agrobiología de Salamanca (IRNASA-CSIC), Apdo. 257, 37071 Salamanca,

Spain.

fDepartamento de Microbiología y Genética, Instituto de Microbiología Bioquímica, Universidad de Salamanca/CSIC, Campus Miguel de Unamuno, 37007 Salamanca, Spain,

Ettrich@greentech.cz

 

Keywords: Photosystem II, Oxygen-evolving complex, PsbP, PsbQ, higher plants

Introduction

Photosynthesis is the process by which the light energy is converted into the chemical energy. It takes place in the thylakoid membrane of higher plants, algae and cyanobacteria, where membrane-embedded, pigment-protein PSII complex performs light-driven oxidation of water with concomitant reduction of the plastoquinone pool. Water splitting is performed in a cluster of four Mn2+ ions located on the lumenal side of photosystem II (PSII) and Ca2+ and Cl- ions are required for optimal activity of this water–oxidase complex. In higher plants the function of Ca2+ and Cl- is modulated by the presence of four extrinsic proteins [1], PsbP, PsbQ [2], PsbO, and PsbR, at the lumenal surface, the so called oxygen-evolving complex. To understand the molecular mechanisms of the oxygen–evolving reaction, an essential prerequisite is the structural knowledge of these proteins and their relative interactions.

Results and Discussion

The structure of recombinant protein PsbP of the oxygen-evolving complex from  Spinacia oleracea was determined at a 1.98 A° resolution by X-ray crystallography [3] and the unresolved parts further refined by Raman and FTIR spectroscopy. spectroscopy gives a unique opportunity to study protein samples in different phases, we report the spectra of PsbP in crystal, solution and as DCDR deposit. For protein preparation the overexpression system Escherichia coli (BL21DE3) transformed by plasmid DNA with gene PsbP (recombinant HisPsbP) was used to produce protein stable in bisTRIS buffer (pH=6,00) in a concentration of 15 mg/ml. Aclearer picture about the location of extrinsic protein in higher plants is emerging from interaction studies between PsbP and PsbQ proteins from spinach, approached in both, theoretical (MD analysis) and experimental level (affinity chromatopgraphy).

References

1.   De Las Rivas J., Heredia P., Roman A.,: Oxygen-evolving extrinsic proteins (PsbO,P,Q,R): Bioinformatics and functional analysis, Biochim. Biophys.Acta 1767 (2007), 575-582

 2.          J. Ristvejova, V. Kopecky, Z. Sovova, M. Balsera, J.B.Arellano, M.Green, R.Ettrich: Structure and dynamics of the N-terminal loop of PsbQ from photosystem II of Spinacea oleracea, Biochem. Biophys. Res. Comm. 345, 287-291, 2006

 3. J.Kohoutová, I. Kutá-Smatanová,  J.Brynda, N.Lapkouski,  J.L. Revuelta., J.B Arellano., R.Ettrich, Crystallization and preliminary crystallographic characterization of the PsbP protein from oxygen-evolving complex of photosystem II from Spinacia oleracea, Acta Cryst. (2009). F65, doi:10.1107/S1744309108040578.

 

Acknowledgements.

We gratefully acknowledge support from Ministry of Education, Youth and Sports of the Czech Republic [MSM6007665808, LC06010]; Academy of Sciences of the Czech Republic [AVOZ60870520];  Grant Agency of the Academy of Sciences of the Czech Republic [KJB101120805 to V.K.]