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Mapping of a copper-binding site on the small CP12 chloroplastic protein of Chlamydomonas reinhardtii using top-down mass spectrometry and site-directed mutagenesis

Abstract : CP12 is a small chloroplastic protein involved in the Calvin cycle that was shown to bind copper, a metal ion involved in modulation of its transition from reduced to oxidized state. In order to describe CP12 copper binding properties, copper-IMAC experiments and site-directed mutagenesis based on computational modeling, were coupled to top-down mass spectrometry (ESI-MS and MSMS). Copper-IMAC experiments allowed the primary characterization of mutation effects upon copper binding. Top-down MSMS experiments carried out under non-denaturing conditions on wild-type and mutant CP12/Cu2+ complexes then allowed fragment ions specifically liganding the copper ion to be determined. Comparison of MSMS datasets defined three regions involved in metal ion binding: residues D16 to D23, D38 to K50 and D70 to E76, with the two first regions containing selected residues for mutation. These data confirmed that copper ligands involved glutamic and aspartic residues in contrast to typical protein copper chelators. We propose that copper might play a role in regulation of CP12 biological activity.
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Submitted on : Friday, April 30, 2010 - 4:15:44 PM
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Jenny Erales, Brigitte Gontero, Julian Whitelegge, Frédéric Halgand. Mapping of a copper-binding site on the small CP12 chloroplastic protein of Chlamydomonas reinhardtii using top-down mass spectrometry and site-directed mutagenesis. Biochemical Journal, Portland Press, 2009, 419 (1), pp.75 - 86. ⟨10.1042/BJ20082004⟩. ⟨hal-00479106⟩

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