Cu(II)-Binding properties of a cytochrome c with a synthetic metal-binding site: His-X3-His in an α-helix

✍ Scribed by Robert J. Todd; Mariana E. Van Dam; Danilo Casimiro; Barry L. Haymore; Dr. Frances H. Arnold

• Book ID: 105358566
• Publisher: John Wiley and Sons
• Year: 1991
• Tongue: English
• Weight: 646 KB
• Volume: 10
• Category: Article
• ISSN: 0887-3585
• DOI: 10.1002/prot.340100209

✦ Synopsis

A metal-binding site consisting of two histidines positioned His-X3-His in an alpha-helix has been engineered into the surface of Saccharomyces cerevisiae iso-1-cytochrome c. The synthetic metal-binding cytochrome c retains its biological activity in vivo. Its ability to bind chelated Cu(II) has been characterized by partitioning in aqueous two-phase polymer systems containing a polymer-metal complex, Cu(II)IDA-PEG, and by metal-affinity chromatography. The stability constant for the complex formed between Cu(II)IDA-PEG and the cytochrome c His-X3-His site is 5.3 x 10(4) M-1, which corresponds to a chelate effect that contributes 1.5 kcal mol-1 to the binding energy. Incorporation of the His-X3-His site yields a synthetic metal-binding protein whose metal affinity is sensitive to environmental conditions that alter helix structure or flexibility.