Characterization of the Metal Binding and Dimerization Properties of the Copper(I) Responsive Repressor Protein from the Enterococcus hirae Copper Homeostasis System

Defense Date


Graduation Date

Fall 1-1-2006


Campus Only

Submission Type


Degree Name



Chemistry and Biochemistry


Bayer School of Natural and Environmental Sciences

Committee Chair

Charles Dameron

Committee Member

Jeffry Madura

Committee Member

Marc Solioz


Large zone size exclusion chromatography, mass spectrometry, affinity resin binding assay


The CopY repressor protein is a central component of the copper homeostasis system of Enterococcus hirae. The protein binds as a homodimer to the promoter DNA of the cop operon, and represses the production of copper control proteins encoded by the genes of the operon. CopY is capable of binding either Zn(II) or Cu(I) in a Cys-x-Cys-xxxx-Cys-x-Cys metal binding site positioned near the C-terminus. Spectrophotometric assays were used to study the mechanism of Cu(I) binding. The results indicate that Zn(II) plays an important role in pre-stabilizing the CopY metal binding site prior to Cu(I) binding. The absence of Zn(II), or the reinsertion after removal, results in improper incorporation of Cu(I) which subsequently promotes the aggregation of CopY. Zn(II) not only affects the Cu(I) binding mechanism, but also contributes to the formation of protein homodimers. Large zone size exclusion chromatography experiments served to measure the equilibrium between the monomeric and dimeric forms of CopY. The absence of Zn(II) in apo-CopY promotes the shifting of the equilibrium toward the monomeric form at low protein concentrations, while the Zn(II)-loaded protein retains the dimeric form. A more thorough investigation of the CopY dimerization mechanism was performed by fusing the 38 C-terminal amino acids of CopY, which contain both the metal binding domain and a sequence of repeated aliphatic amino acids, to a small, monomeric protein. The ability of the CopY truncate to facilitate dimerization was assessed by electrospray ionization mass spectrometry, an affinity resin binding assay, and large zone size exclusion chromatography. The results indicate that the CopY truncate promotes dimerization, and that association is dependent on both metal binding and hydrophobic interactions through the aliphatic amino acids. Measurement of the monomer-dimer equilibrium by large zone chromatography indicates that removal of metal weakens the dimer interaction to a greater extent than does the mutation of the aliphatic amino acids to hydrophilic residues. The simultaneous loss of both the metal binding and the aliphatic residues appears to eliminate dimerization. The elucidation of the CopY dimerization mechanism advances the potential use of the C-terminal sequence as a biotechnological tool to control association or dissociation of molecules.





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