Characterization of the recombinant copper chaperone (CCS) from the plant Glycine (G.) max.
The goal of the present work was to characterize the recombinant copper chaperone (CCS) from soybean. Very little is known about plant copper chaperones, which makes this study of current interest, and allows for a comparison with the better known homologues from yeast and humans. To obtain sizeable amounts of pure protein suitable for spectroscopic characterization, we cloned and overexpressed the G. max CCS chaperone in E. coli in the presence of 0.5 mM CuSO(4) and 0.5 mM ZnSO(4) in the broth. A pure protein preparation was obtained by using two IMAC steps and pH gradient chromatography. Most of the proteins were obtained as apo-form, devoid of copper atoms. The chaperone showed a high content (i.e., over 40%) of loops, turns and random coil as determined both by circular dichroism and homology modelling. The homology 3-D structural model suggests the protein might fold in three structural protein domains. The 3-D model along with the primary structure and spectroscopic data may suggest that copper atoms occupy the two metal binding sites, MKCEGC and CTC, within the N-terminal domain I and C-terminal domain III, respectively. But only one Zn-binding site was obtained spectroscopically.
Estación Experimental de Aula Dei, Consejo Superior de Investigaciones Científicas, Carretera Montañana 1005, E-50059 Zaragoza, Spain. sagasti@eead.csic.es
Sara
S
Sagasti
Inmaculada
I
Yruela
Maria
M
Bernal
Maria A
MA
Lujan
Susana
S
Frago
Milagros
M
Medina
Rafael
R
Picorel
D000595:Amino Acid Sequence; D001665:Binding Sites; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D003300:Copper / Q000737:chemistry* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D030262:Soybean Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013025:Glycine max / Q000235:genetics / Q000378:metabolism*; D015032:Zinc / Q000737:chemistry / Q000378:metabolism
D018832:Molecular Chaperones; D011994:Recombinant Proteins; D030262:Soybean Proteins; D003300:Copper; D015032:Zinc