Incorporation of histidine-rich metal-binding sites onto small protein scaffolds

Abstract

Many histidine-rich sites in proteins bind transition metal ions such as Zn, natively. Such sites can encourage proper protein folding or allow access to enzymatic capabilities such as hydrolysis. Ru(II) and Tc(I) also bind to aromatic amines providing access to unique chemistries not observed in biology. Ru(II) complexes have shown efficacy in fighting cancer and catalysis, while Tc complexes are used in radioimaging. To incorporate such metal-ions’ activities into proteins, several mutants have been designed to bind Zn, Ru, and Tc by introducing three histidines onto their surfaces. The first design, Z0, utilized a chimeric approach by substituting a turn in engrailed homeodomain for the superimposable Zn-binding loop of astacin. In the second design, 3HT-C, three histidine residues were incorporated into the N-terminus of the Trp-cage. The final scaffold, ubiquitin, was used to make two mutants: 3HIU with a 3-histidine containing loop inserted between residues 9 and 10, and 3HPU with 3histidine point mutations near residues 35-38. Z0 proved unstable due to incorporation of a hydrophobic patch onto its surface and was not able to be isolated in sufficient quantities for study. However, the other proteins were stable and soluble. Zn-binding by 3HT-C was investigated by intrinsic tryptophan fluorescence quenching, circular dichroism, and RP-HPLC. Binding by 3HIU and 3HPU was studied by CD. All designed proteins bind to Zn with Kd values in the micromolar range. 3HIU and 3HPU were further studied for their ability to bind Ru(tacn) complexes. While addition of Rucomplexes caused oligomerization to various extents depending upon reaction conditions, homogeneous Ru-protein monomers were purified by a combination of size-exclusion, cation-exchange and immobilized-metal affinity chromatographies. Ru-binding was confirmed by ESI-MS, and structural integrity was investigated by CD. Results indicate that Ru(tacn) complexes can be bound to surface binding sites in proteins without disruption of structure, opening the door for the study of catalysis in a protein context.