Mapping of a copper-binding site on the small CP12 chloroplastic protein of Chlamydomonas reinhardtii using top-down mass spectrometry and site-directed mutagenesis.
CP12 is a small chloroplastic protein involved in the Calvin cycle that was shown to bind copper, a metal ion that is involved in the transition of CP12 from a reduced to an oxidized state. In order to describe CP12's copper-binding properties, copper-IMAC experiments and site-directed mutagenesis based on computational modelling, were coupled with top-down MS [electrospray-ionization MS and MS/MS (tandem MS)]. Immobilized-copper-ion-affinity-chromatographic experiments allowed the primary characterization of the effects of mutation on copper binding. Top-down MS/MS experiments carried out under non-denaturing conditions on wild-type and mutant CP12-Cu(2+) complexes then allowed fragment ions specifically binding the copper ion to be determined. Comparison of MS/MS datasets defined three regions involved in metal ion binding: residues Asp(16)-Asp(23), Asp(38)-Lys(50) and Asp(70)-Glu(76), with the two first regions containing selected residues for mutation. These data confirmed that copper ligands involved glutamic acid and aspartic residues, a situation that contrasts with that obtaining for typical protein copper chelators. We propose that copper might play a role in the regulation of the biological activity of CP12.
Erales
Jenny
J
Laboratoire Enzymologie des Complexes Supra-moléculaires, BIP, UPR 9036-IFR 88, CNRS-Universités Aix Marseille I et III, BP 71, 31 Chemin Joseph Aiguier, 13402 Marseille Cédex 20, France.
Gontero
Brigitte
B
Whitelegge
Julian
J
Halgand
Frédéric
F
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D016825:Chlamydomonas reinhardtii / Q000235:genetics / Q000378:metabolism*; D002736:Chloroplasts / Q000378:metabolism*; D002942:Circular Dichroism; D003300:Copper / Q000378:metabolism*; D015151:Immunoblotting; D013058:Mass Spectrometry; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010788:Photosynthesis / Q000502:physiology; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
D010940:Plant Proteins; D003300:Copper