Characterization of a small metal binding protein from Nitrosomonas europaea.
A small metal-binding protein (SmbP) with no known similarity to other proteins in current databases was isolated and characterized from the periplasm of Nitrosomonas europaea. The primary structure of this small (9.9 kDa) monomeric protein is characterized by a series of 10 repeats of a seven amino acid motif and an unusually high number of histidine residues. The protein was isolated from N. europaea with Cu(II) bound but was found to be capable of binding multiple equivalents of a variety of divalent and trivalent metals. The protein was overexpressed in Escherichia coli and used for the study of its metal-binding properties by UV/vis, circular dichroism (CD), and electron paramagnetic resonance (EPR) spectroscopy and equilibrium dialysis and isothermal titration calorimetry. The protein was found to bind up to six Cu(II) atoms with dissociation constants of approximately 0.1 microM for the first two metal ions and approximately 10 microM for the next four. Binding of Cu(II) resulted in spectroscopic features illustrating two distinctive geometries, as determined by EPR spectroscopy. The levels of SmbP in the periplasm were found to increase by increasing the levels of copper in the growth media. This protein is proposed to have a role in cellular copper management in the ammonia-oxidizing bacterium N. europaea.
Barney
Brett M
BM
Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona 85287, USA.
LoBrutto
Russell
R
Francisco
Wilson A
WA
D000595:Amino Acid Sequence; D001422:Bacterial Adhesion; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D001665:Binding Sites; D002413:Cations, Divalent / Q000378:metabolism; D002942:Circular Dichroism; D003300:Copper / Q000737:chemistry / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D008667:Metalloproteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D043370:Nitrosomonas europaea / Q000737:chemistry* / Q000378:metabolism; D033902:Periplasmic Binding Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D017433:Protein Structure, Secondary; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D001426:Bacterial Proteins; D002413:Cations, Divalent; D008667:Metalloproteins; D033902:Periplasmic Binding Proteins; D003300:Copper