Use of metal-binding de novo-designed alpha-helical peptides in the study of metalloprotein structures.

Abstract

A family of de novo-designed $\alpha$-helical peptides were created to study how metals and polypeptides interact when forming metalloprotein complexes. The amphiphilic peptide Tri, based on the repeat (L K A L E E K), was designed to form coiled coils. Leu in a and d positions were used for hydrophobic packing, and Glu/Lys were inserted for salt bridging. To generate a metal-binding site, single Cys substitutions were made for Leu, generating a (s-Cys)$\rm\sb{n}$ site where n is the peptide's aggregation state. All peptides showed concentration dependence to their helicities, indicating that aggregation occurred and was required for helical secondary structure. Sedimentation equilibrium and gel filtration showed that a pH-dependent aggregation change occurred for Tri, with 2-helix and 3-helix bundles predominant at pHs 2.5 and 8.5, respectively. Cys-substituted derivatives showed a shift in the 2-helix/3-helix equilibrium, with the 2-helix aggregate stable to higher pHs. A pH dependence was also observed in the peptide stability towards chemical denaturation, with denaturation midpoints largest at pH 2.5 and decreasing with increasing pH, indicating that the 2-helix aggregates were more stable than 3-helix bundles. Substitution of Cys for Leu lowered the stability of the peptide towards denaturation. The Hg-binding properties of the Cys-derivatives were studied using UV titrations, $\sp{199}$Hg-NMR, EXAFS and sedimentation. The a and d substitutions proved to be distinct in their coordination chemistries with Hg(II). For a substitutions, 3-coordinate Hg(S-Cys)$\sb3$ complexes could be formed at pH 8.5 when a 1:3 Hg:peptide ratio was used, whereas at pH 5.5 or with a 1:2 Hg:peptide ratio, Hg(S-Cys)$\sb2$ complexes were produced. In contrast, d substituants only generated Hg(S-Cys)$\sb2$ at all pHs and Hg/peptide ratios. Based on modelling, this difference is due to the orientations relative to the superhelical axis, with a pointing in towards the axis and d pointing outward. Addition of Hg$\sp{2+}$ to all Cys-derivatives increased the stability of the peptide aggregates towards chemical denaturation substantially, with an increase in the observed denaturation midpoint of at least 3.5M Gua-HCl. These peptides, with their ability to control ligand geometry as well as aqueous exposure of the binding site, are an important step toward understanding de novo-designed metallopeptides.