Erika Bladholm - Incorporation of the red copper nitrosocyanin binding loop into blue copper azurin

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  Publication Details (including relevant citation   information):

  Berry, S. M.;   Bladholm, E. L.; Mostad, E. J.; Schenewerk, A.   R., J. Biol. Inorganic   Chemistry, 2011, 16, (3), 473-480



    Loop-directed mutagenesis was applied to the blue copper     protein azurin to replace its copper binding loop with that     from the red copper protein nitrosocyanin. A ten amino acid     long loop that provides three of the four copper ligands from     nitrosocyanin was incorporated into azurin to make a variant     called NC-azurin. The chimeric protein displayed a red color,     and UV–vis absorption and EPR spectra that closely resembled     those of the loop parent, nitrosocyanin. We added the fourth     ligand from nitrosocyanin into NC-azurin, a     carboxylate-containing amino acid, but the proteins had altered     stability and spectroscopic properties that did not resemble     those of either parent copper protein. The loop alone, however,     was enough to impart red copper site characteristics to the     NC-azurin protein. Finally, the reduction potential of the     variant was found to be between the reduction potentials of the     parent proteins and about 50 mV below that of wild-type azurin.  

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