Stability and nickel binding properties of peptides designed as scaffolds for the stabilization of Ni(II)-Fe(4)S(4) bridged assemblies.

Helix-loop-helix peptides containing 63 residues (HC(4)H(2), HC(4)HC, HC(5)H), designated by their sequence and content of histidyl (H) and cysteinyl (C) residues, have been previously synthesized for the purpose of stabilizing certain bridged metal sites in proteins. These peptides bind one Fe(4)S(4) cluster by means of a ferredoxin tricysteinyl consensus sequence and an additional Cys residue, and one Ni(II) atom (HC(4)H(2), HC(5)H) in predesigned binding sites. In this investigation, the apopeptides and their Fe(4)S(4) derivatives are shown to be relatively stable to unfolding by guanidine hydrochloride, indicating stability of secondary structure. With this property demonstrated, Ni(II) binding equilibria have been evaluated in the terms of site-specific (Scatchard model) and stepwise (stoichiometric) binding constants. Two peptides were designed to have preformed CysHis(3) (HC(4)H(2)) and Cys(2)His(2) (HC(5)H) binding sites. The data indicate one strong binding site in each peptide with preferred binding constants k(1)=4.4x10(5) M(-1)(HC(4)H(2)) and 2.7x10(5) M(-1)(HC(5)H). Based on X-ray absorption spectroscopic data, these binding steps are associated with the formation of the desired coordination units Ni(II)CysHis(3) and Ni(II)Cys(2)His(2). For peptide HC(4)HC, k(1)=2.5x10(5) M(-1), but the binding site could not be fully identified. Collective evidence from this and prior investigations supports the presence of the bridged assemblies Ni(II)-(mu(2)-S x Cys)-[Fe(4)S(4)], stabilized by a scaffolding effect in peptides HC(4)H(2) and HC(5)H. The assembly Ni(II)-X-[Fe(4)S(4)] is the minimal structure of the A-Cluster of carbon monoxide dehydrogenase adduced from spectroscopic evidence; bridge X is currently unidentified. These results suggest that de novo designed peptides may serve as scaffolds for the construction of native bridged sites in proteins. Electronic supplementary material to this paper can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00775-001-0320-4.