Abstract
Understanding the structural determinants for metal ion coordination in metalloproteins is a fundamental issue for designing metal binding sites with predetermined geometry and activity. In order to achieve this, we report in this paper the design, synthesis and metal binding properties of METP3, a homodimer made up of a small peptide, which self assembles in the presence of tetrahedrally coordinating metal ions. METP3 was obtained through a redesign approach, starting from the previously developed METP molecule. The undecapeptide sequence of METP, which dimerizes to house a Cys4 tetrahedral binding site, was redesigned in order to accommodate a Cys2His2 site. The binding properties of METP3 were determined toward different metal ions. Successful assembly of METP3 with Co(II), Zn(II) and Cd(II), in the expected 2:1 stoichiometry and tetrahedral geometry was proven by UV-visible spectroscopy. CD measurements on both the free and metal-bound forms revealed that the metal coordination drives the peptide chain to fold into a turned conformation. Finally, NMR data of the Zn(II)-METP3 complex, together with a retrostructural analysis of the Cys-X-X-His motif in metalloproteins, allowed us to define the model structure. All the results establish the suitability of the short METP sequence for accommodating tetrahedral metal binding sites, regardless of the first coordination ligands.
Highlights
Metalloproteins employ a limited number of metal ions to accomplish countless different functions, including electron transfer, signal transduction and enzymatic catalysis [1,2,3].To achieve this incredible variety, a wide array of protein sites has been selected over billions of years of evolution, each purposed towards a specific task [4]
Protein folding occurs upon metal ion coordination, while in others, tight metal binding is assured by the protein, which imparts its own structural preferences to the metal ion [3]
The aim of this work was to assess whether a short peptide sequence could be used as scaffold for developing metal binding sites capable of binding different metal ions with identical coordination geometries
Summary
Metalloproteins employ a limited number of metal ions to accomplish countless different functions, including electron transfer, signal transduction and enzymatic catalysis [1,2,3]. To achieve this incredible variety, a wide array of protein sites has been selected over billions of years of evolution, each purposed towards a specific task [4]. The trigonal His Cys coordination site observed in azurin is not optimal for either the Cu(II) or the Cu(I) oxidation states, and only minimal structural changes occur upon redox cycling.
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