Cu(II) coordination in Proteins

Coordination Features and Affinity of the Cu2+ site in the alpha-Synuclein Protein of Parkinson’s Disease

Dudzik, C.G.; Walter, E.D. and Millhauser, G.L.

Biochemistry ASAP

Alpha-synuclein proteins, concentrated in neurons in the human body, are composed of mostly hydrophobic residues. The hydrophobicity of the protein results in frequent aggregation or ‘clumping together’ of multiple alpha-syn proteins to form large fibril structures. These protein aggregates have been linked to the neurological disorder Parkinson’s disease. It has not been shown that these aggregates are a facilitator in Parkinson’s disease progression, but the increased levels of alpha-syn in Parkinson’s patients provides an intriguing subject for research. Alpha-syn has previously shown affinity for copper(II), but without evidence of binding ratios (stoichiometery), binding site, and binding affinity. The authors of this paper relied heavily on Electron Paramagnetic Resonance (EPR) to characterize the binding of copper to the protein’s amino acids. This technique takes advantage of the unpaired electrons of the Cu2+ (which has 9 electrons in the d orbital) to gain insight about the environment of the unpaired electron. The EPR data suggests a three nitrogen one oxygen coordination environment for the copper ion.

Based on this data and previously reported experiments, the authors assigned the copper binding site as shown above.

In addition, the authors used EPR to find the equilibrium constant for binding (Kd). The constant was determined by adding copper binding agent to the protein-copper sample. The copper binding agent (oxidized glutathione and pentaglycine) will sequester copper which will change the EPR signal seen (because the environment around copper ion will change). Based on the amount of change seen the spectrum, the binding constant of the copper to the protein was calculated. The binding affinity experiments demonstrated that one copper ion is bound tightly to the protein. Histidine 50 coordination was confirmed through mutagenesis studies (changing the His50 to a non-binding alanine residue).

In summation, this work demonstrates one method that can be employed to determine if a metal ion is coordinated to a protein chain. Although, this work focused almost exclusively on EPR methods, other methods such as UV-vis, Mossbauer, and X-ray crystallography are often suitable when determining the possibility of metal-protein interactions.

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