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 wildtype azurin.
Bibliographical noteFunding Information:
Acknowledgments The authors thank the Swenson Family Foundation, UM-GIA, and the Camille and Henry Dreyfus Foundation for funding and student stipends. We thank Mark J. Nilges of the Illinois EPR Research Center for assistance gathering EPR data. Finally, we would like to thank John H. Richards, California Institute of Technology, and Yi Lu, University of Illinois at Urbana-Champaign, for the kind gift of the azurin gene.
Copyright 2011 Elsevier B.V., All rights reserved.
- Blue copper protein
- Loop-directed mutagenesis
- Red copper protein