Abstract
State transitions in cyanobacteria are physiological adaptation mechanisms that change the interaction of the phycobilisomes with the photosystem I and photosystem II core complexes. This mechanism is essential for cyanobacteria at low light intensities. Previous studies of cyanobacteria have identified a gene named rpaC, which appears to be specifically required for state transitions. The gene product of rpaC is very probably a transmembrane protein that is a structural component of the phycobilisome-photosystem II supercomplex. However, the physiological role of RpaC protein is unclear.
Here we report the construction of an expression system that enables high production of fusion protein TrxHisTagSTag-RpaC, and describe suitable conditions for purification of this insoluble protein at a yield of 3 mg per 1 dm3 of bacterial culture. Cleavage with HRV 3C protease to remove the TrxHisTagSTag portion resulted in low yields of RpaC-protein (∼ 30 µg/dm3 of bacterial culture), therefore the applicability to structural studies was tested for the fusion protein only. Several preliminary conditions for crystallization of TrxHisTagSTag-RpaC were set up under which microcrystals were obtained. This set of conditions will be a good starting point for optimization in future crystallization experiments. TrxHisTagSTag-RpaC protein may prove useful in biochemical studies where the small size of RpaC protein is limiting the investigation of interactions with significantly larger parts of the photosynthetic apparatus. Furthermore, the purification procedure described here might also be applied to the production and purification of other small membrane proteins for biochemical and structural studies.
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References
Allen, J.F., Bennett, J., Steinback, K.E., Arntzen, C.J.: Chloroplast protein phosphorylation couples plastoquinone redox state to distribution of excitation energy between photosystems. — Nature 291: 25–29, 1981.
Allen, J.F. and Forsberg, J.: Molecular recognition in thylakoid structure and function. — Trends Plant Sci. 6: 317–326, 2001.
Bonaventura, C. and Myers, J.: Fluorescence and oxygen evolution from Chlorella pyrenoidosa. — Biochim. Biophys. Acta 189: 366–383, 1969.
Depege, N., Bellafiore, S., Rochaix, J.D.: Role of chloroplast protein kinase Stt7 in LHCII phosphorylation and state transition in Chlamydomonas. — Science 299: 1572–1575, 2003.
Emlyn-Jones, D., Ashby, M.K., Mullineaux, C.W.: A gene required for the regulation of photosynthetic light harvesting in the cyanobacterium Synechocystis 6803. — Mol. Microbiol. 33: 1050–1058, 1999.
Fork, D.C., Satoh, K.: State I-state-II transitions in the thermophilic blue-green-alga (cyanobacterium) Synechococcus lividus. — Photochem. Photobiol. 37: 421–427, 1983.
Horton, P., Allen, J.F., Black, M.T., Bennett, J.: Regulation of phosphorylation of chloroplast membrane polypeptides by the redox state of plastoquinone. — FEBS Lett. 125: 193–196, 1981.
Horton, P., Black, M.T.: Activation of adenosine 5′ triphosphate-induced quenching of chlorophyll fluorescence by reduced plastoquinone. The basis of state-I-state-II transitions in chloroplasts. — FEBS Lett. 119: 141–144, 1980.
Joshua, S., Mullineaux, C.W.: The rpaC gene product regulates phycobilisome-photosystem II interaction in cyanobacteria. — Biochim. Biophys. Acta 1709: 58–68, 2005.
Kaneko, T., Nakamura, Y., Wolk, C.P., Kuritz, T., Sasamoto, S., Watanabe, A., Iriguchi, M., Ishikawa, A., Kawashima, K., Kimura, T., Kishida, Y., Kohara, M., Matsumoto, M., Matsuno, A., Muraki, A., Nakazaki, N., Shimpo, S., Sugimoto, M., Takazawa, M., Yamada, M., Yasuda, M., Tabata, S.: Complete genomic sequence of the filamentous nitrogen-fixing Cyanobacterium anabaena sp. strain PCC 7120. — DNA Res. 8: 205–213, 2001.
Mueller-Dieckmann, J.: The open-access high-throughput crystallization facility at EMBL Hamburg. — Acta Crystallogr. 62: 1446–1452, 2006.
Mullineaux, C.W.: Excitation energy transfer from phycobilisomes to photosystem I in a cyanobacterial mutant lacking photosystem II. — Biochim. Biophys. Acta 1184: 71–77, 1994.
Mullineaux, C.W., Emlyn-Jones, D.: State transitions: an example of acclimation to low-light stress. — J. Exp. Bot. 56: 389–393, 2005.
Mullineaux, C.W., Tobin, M.J., Jones, G.R.: Mobility of photosynthetic complexes in thylacoid membranes. — Nature 390: 421–424, 1997.
Murata, N.: Control of excitation transfer in photosynthesis. I. Light-induced change of chlorophyll a fluorescence in Porphyridium cruentum. — Biochim. Biophys. Acta 172: 242–251, 1969.
Rakhimberdieva M.G., Boichenko V.A., Karapetyan N.V., Stadnichuk I.N.: Interaction of phycobilisomes with photosystem II dimmers and photosystem I monomers and trimers in the cyanobacterium Spirulina platensis. — Biochemistry 40: 15780–15788, 2001.
Schägger, H., von Jagow, G.: Tricine-sodium sulfate-polyacrylamide gel electroforesis for the separation of proteins in the range from 1 to 100 kDa. — Anal. Biochem. 166: 368–379, 1987.
van Thor, J.J., Mullineaux, C.W., Matthijs, H.C.P., Hellingwert, K.J.: Light harvesting and state transitions in cyanobacteria. — Bot. Acta 111: 430–443, 1998.
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Acknowledgements: Authors thank to Dr. Rüdiger Ettrich for his scientific help and language corrections. Support from the Ministry of Education, Youth and Sports of the Czech Republic (MSM6007665808), the Academy of Sciences of the Czech Republic (AVOZ60870520, KJB500870701), and the Grant agency of the Czech Republic (206/07/0917). We thank EMBL for access to the X11 beamline at the DORIS storage ring of DESY in Hamburg.
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Cséfalvay, E., Lapkouski, M. & Komárek, O. Expression, purification and preliminary crystallization study of RpaC protein from Synechocystis sp. PCC6803. Photosynthetica 47, 355–362 (2009). https://doi.org/10.1007/s11099-009-0056-x
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DOI: https://doi.org/10.1007/s11099-009-0056-x