Miniaturized metalloproteins: Application to iron-sulfur proteins

The miniaturization process applied to rubredoxins generated a class of pep tide-based metalloprotein models, named METP (miniaturized electron transfe r protein). The crystal structure of Desulfovibrio vulgaris rubredoxin was selected as a template for the construction of a tetrahedral (S-gamma-Cys)( 4) iron-binding site. Analysis of the structure showed that a sphere of 17 Angstrom in diameter, centered on the metal, circumscribes two unconnected approximately Ct symmetry related beta -hairpins, each containing the -Cys- (Aaa)(2)-Cys-sequence. These observations provided a starting point for the design Of an undecapeptide, which self assembles in the presence of tetrah edrally coordinating metal ions. The METP peptide was synthesized in good y ield by standard methodologies. Successful assembly of the METP peptide wit h Co(II), Zn(II), Fe(II/III), in the expected 2:1 stoichiometry, was proven by UV-visible and circular dichroism spectroscopies. UV-visible analysis o f the metal complexes indicated the four Cys ligands tetrahedrally arrange around the metal ion, as designed. Circular dichroism measurements on both the free and metal-bound forms revealed that the metal coordination drives the peptide chain to fold into a turned conformation. NMR characterization of the Zn(II)-METP complex fully supported the structure of the designed mo del. These results prove that METP reproduces the main features of rubredox in.