Crystallization of dimers of the manganese-stabilizing protein of Photosystem II |
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Authors: | Anati Rina Adir Noam |
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Institution: | (1) Department of Chemistry and Institute of Catalysis, Science and Technology, Technion – Israel Institute of Technology, Technion City, Haifa, 32000, Israel;(2) Department of Chemistry and Institute of Catalysis, Science and Technology, Technion – Israel Institute of Technology, Technion City, Haifa, 32000, Israel |
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Abstract: | The manganese-stabilizing protein (MSP) of Photosystem II was purified from spinach photosynthetic membranes. The MSP was
crystallized in the presence of calcium. Despite the apparent purity of the isolated protein, the crystals grew to only about
0.05 mm in their largest dimension. The MSP was analyzed to identify possible sources of protein heterogeneity that could
hinder crystal growth. Tandem reverse-phase HPLC/ electronspray ionization mass spectrometry analysis of the MSP showed a
major peak and four smaller peaks. All five peaks had molecular masses of 26 535, as expected for mature MSP, indicating the
absence of heterogeneities due to covalent modifications. MALDI mass spectroscopy was utilized to identify heterogeneities
in the MSP oligomeric state. These measurements showed that purified MSP in solution is a mixture of monomers and dimers,
while solubilized MSP crystals contained only dimers. Size-exclusion chromatography and dynamic light scattering were used
to probe the effect of the crystallization conditions on the MSP. Size-exclusion chromatography of concentrated MSP showed
the presence of aggregates and monomers, while dilute MSP contained monomers. Dynamic light scattering experiments in the
absence, or in the presence of 10–50 mM or 100 mM calcium, yielded calculated molecular mass values of 34 kDa, 48 kDa and
68 kDa, respectively. These changes in the observed molecular mass of the MSP could have been caused by the formation of dimers
and higher oligomers and/or significant conformational changes. Based on the results reported in this study, a model is presented
which details the effect of oligomeric heterogeneity on the inhibition of MSP crystal growth.
This revised version was published online in June 2006 with corrections to the Cover Date. |
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Keywords: | dynamic light scattering mass spectrometry oxygen evolution photosynthesis protein crystallization protein structure |
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