Recombinant Protein Stabilization through Engineered Metal-Chelating Sites

Abstract

Simple metal-chelating sites incorporated into common elements of secondary structure located on a protein surface offer a powerful and general strategy for stabilizing recombinant proteins. By binding with higher affinity to the native state of the protein, a metal ion can shift the folding/unfolding equilibrium toward the native state. To demonstrate this approach, we have engineered metal-chelating sites consisting of pairs of histidine residues into Saccharomyces cerevisiae iso-1-cytochrome c. 1 mM Cu(II) complexed to iminodiacetate stabilizes the cytochrome c variants by 1-2 kcal/mol, as determined by guanidinium chloride-induced unfolding. The increase in the free energy for unfolding is equal to that calculated from the preferential binding of the metal ion to the native protein. The Cu(II) affinities of di-histidine sites introduced in α-helices of bovine somatotropin indicate a potential for increasing stability by as much as 3.5 kcal/mol with a single di-histidine site. Chelating sites are easily introduced into surface α-helices and β-sheets while introducing minimal or no disruption of the native structure or biological function.

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