Miniaturized metalloproteins: Application to iron–sulfur proteins

Abstract

The miniaturization process applied to rubredoxins generated a class of peptide-based metalloprotein models, named METP (miniaturized electron transfer protein). The crystal structure of Desulfovibrio vulgaris rubredoxin was selected as a template for the construction of a tetrahedral (S ␥ -Cys)4 iron-binding site. Analysis of the structure showed that a sphere of 17 Å in diameter, centered on the metal, circumscribes two unconnected approximately C2 symmetry related ␤-hairpins, each containing the -Cys-(Aaa)2-Cyssequence. These observations provided a starting point for the design of an undecapeptide, which self assembles in the presence of tetrahedrally coordinating metal ions. The METP peptide was synthesized in good yield by standard methodologies. Successful assembly of the METP peptide with Co(II), Zn(II), Fe(II͞III), in the expected 2:1 stoichiometry, was proven by UV-visible and circular dichroism spectroscopies. UV-visible analysis of 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 model. These results prove that METP reproduces the main features of rubredoxin. Miniaturized proteins are peptide-based synthetic models of natural macromolecular systems. They contain a minimum set of constituents necessary for an accurate reconstruction of defined structures and for a fine-tuned reproduction of defined functions (1, 2). Their intermediate size between low molecular weight compounds and protein mutants makes miniaturized proteins suitable for structure-function relationship studies: they are simple enough to avoid ambiguities of interpretation associated with large proteins, and they provide sufficient size and chemical diversity to allow the construction of active sites.The miniaturization process can be rationally organized, once a structural knowledge of the parent natural system to be miniaturized is available. It is necessary to define (i) the type and number of constituents to be assembled, (ii) the structure to be reconstructed, and (iii) the function to be reproduced. These aspects are strictly related. A larger number of constituents might be necessary for a more precise three-dimensional reconstruction and for a more specialized function.Metalloproteins are well suited to miniaturization. The metal center represents a pivotal point where spheres of variable diameters that circumscribe part of the protein are centered: the larger the diameter of the sphere, the larger the number of constituents included in the model. Comparative studies, in terms of structure and function, of a set of protein models, defined by spheres of variable diameters, will allow better understanding of how the protein matrix modulates the unique features of the metal center, including electronic, redox, and ...

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De NovoDesign of Helical Bundles as Models for Understanding Protein Folding and Function

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