An Engineered Azurin Variant Containing a Selenocysteine Copper Ligand

Citations Over TimeTop 11% of 2002 papers

Abstract

Modulating the properties of proteins through de novo design or redesign of existing proteins has been a longstanding goal in protein chemistry. Over the past two decades, site-directed mutagenesis has been a powerful tool to probe the role of certain residues and to fine-tune the activity of proteins. A limitation of this approach has been the accessibility of only a restricted number of functional groups through the 20 amino acids in the genetic code. The more recent technique of expressed protein ligation (EPL) provides an alternative route that allows efficient incorporation of nonnatural residues into proteins. We report here the preparation and spectroscopic characterization of an azurin variant in which a cysteine ligand to the blue copper center has been replaced by EPL with selenocysteine (Sec). This reports marks the first time that selenocysteine is artificially incorporated into the active site of a metalloprotein. The variant displays a significantly increased A(parallel) (from 56 to 104 G) and red-shifted CT band (from 625 to 677 nm), while maintaining the general type 1 copper characteristics, including similarity in reduction potentials. This study illustrates that iso-structural substitution using EPL can fine-tune the structural and functional properties of a metal-binding site without loss of most of its characteristics. Further spectroscopic and X-ray crystallographic studies of this and other EPL variants will provide new insights into the fine-control of the structure and function of metalloproteins.

Related Papers

• → Gene synthesis, expression, and mutagenesis of the blue copper proteins azurin and plastocyanin.(1991)60 cited
• → Structural, dynamical and functional aspects of the inner motions in the blue copper protein azurin(2006)22 cited
• → Structural heterogeneity of blue copper proteins: an EPR study of amicyanin and of wild-type and Cys3Ala/Cys26Ala mutant azurin(2001)8 cited
• → Structure and Electron Transfer Reactions of Blue Copper Proteins(1977)4 cited
• → Understanding the blue copper proteins(1993)5 cited