A New Approach to Protein Design: Grafting of a Buried Transition Metal Binding Site into Escherichia coli Thioredoxin
De Yonker, James Daniel
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The computer program Dezymer is used to insert a "blue copper" binding site into a protein of known structure. This paper examines an approach designed to introduce a copper-binding site into Echerichia coli thioredoxin. The design program finds a constellation of backbone sites that can accommodate the "blue-copper" site in such a way that conserves the backbone conformation of native thioredoxin. These mutations create a new binding site specific for transition metals in a hydrophobic core. Difficulties in the reconstitution of copper into the grafted site suggests factors other than surface binding sites are influential. It appears that the cysteine, which is part of the grafted site, must be discouraged from disulfide dimerization. Using copper(II) complexes may help inhibit disulfide formation and allow copper(II) to bind to the desired site. However, DEZYMER also predicted a permuted geometry of the blue copper site which would prelude this reaction.