Switching the chirality of the metal environment alters the coordination mode in designed peptides.

De novo metallopeptide design contributes significantly to the understanding of protein folding, protein–protein interactions, and metal-ion sites in biology. Within this rubric, studies in our research group have been focused on the biochemistry of Cd thiolate sites. A class of peptides based on the parent peptide TRI, Ac-G(LKALEEK)4G-NH2, was designed to assemble in aqueous solution into amphiphilic a-helices. These peptides aggregate to form three-stranded coiled coils above pH 5.5. Substitutions can be made to the interior of these coiled coils to generate metal-binding sites, potentially with adjacent cavities. One benefit of de novo design is that we are no longer limited to the 20 naturally coded amino acids. We demonstrated by two different strategies that it is possible to create a coordinatively unsaturated trigonal-planar CdS3 site by incorporating nonprotein amino acids into the peptide sequence. The first approach was to replace the coordinating l-cysteine (l-Cys) residue with the bulkier nonprotein analogue l-penicillamine (l-Pen), in which the b-methylene hydrogen atoms have been replaced with larger methyl groups. Our second approach was to modify the chirality of the second-coordination-sphere ligands directly above the metal-binding plane (l-Leu! d-Leu). In this way, the Leu side chain was reoriented towards the C terminus and the metal-binding site. Presumably, this modification sterically inhibits the binding of a fourth ligand to the Cd center. Although these accomplishments were significant, we soon realized that we neither understood how l-Pen was capable of enforcing a CdS3 coordination sphere, nor how the introduction of a single layer of d-amino acids in the interior of a coiled coil composed of l-amino acids would affect the structure. We also realized that we could now assess how metal binding would be perturbed by combining increased steric bulk and alternate chirality within the same amino acid. One might expect that the simple replacement of an l with a d ligand would lead to the same coordination geometry with an inverted stereochemical configuration; however, since these peptides are diastereopeptides, this stereochemical modification is occurring within a sea of l-amino acids. These issues could be best addressed by performing comparative spectroscopic and structural studies on three-stranded coiled coils containing either l-Pen or d-Pen (see Figure S1 in the Supporting Information). We chose to perform experiments on the related peptide Coil Ser (CS), Ac-E WEALEKK LAALESK LQALEKK LEALEHG-NH2, with land d-Pen substituted at the 16-position (CSL16l-Pen and CSL16d-Pen), as Coil Ser behaves similarly to the TRI family, with which the majority of studies have been carried out, yet has proven to be more readily crystallizable. First, we explored the interactions of these peptides in aqueous solution with Cd. Cd NMR spectroscopy is a powerful tool for determining the coordination environment of Cd. The Cd NMR spectrum for Cd bound to the l-amino acid analogue of Pen, CSL16l-Pen, shows a single resonance at d= 683 ppm. This signal is almost identical to that reported previously for the TRI analogue (d= 684 ppm) and thus confirms that Cd is bound as a trigonal-planar three-coordinate species. For the d-Pen analogue, a resonance of weaker intensity was observed at d= 557 ppm, which is well outside the range associated with CdS3 (d= 680– 700 ppm) and more consistent with an S2O2-type (or possibly S3O-type) first-coordination-sphere environment (see Figure S2 in the Supporting Information). The binding of Cd to these constructs was also monitored by UV/Vis spectroscopy and was once again found to be different for the land d-Pen analogues (see Figure S3 in the Supporting Information). The characteristic ligand-to-metal charge transfer (LMCT) band for Cd bound to l-Pen as Cd(CSL16l-Pen)3 is similar (lmax= 227 nm, e227= 36000m 1 cm ) to that reported for Cd(TRIL16l-Pen)3 . In contrast, an absorption band at lmax 210 nm was observed for solutions containing Cd in the presence of CSL16d-Pen at pH 9.6, a pH value at which we generally expect Cd to be fully bound. To determine the pH dependence associated with Cd binding, we monitored absorbance as a function of pH value. The resulting pH titration curves (see Figure S4 in the Supporting Information) reveal that Cd binds to CSL16lPen with a high pKa2 value (16.3), which is consistent with the behavior of Cd binding to a three-coordinate site. However, no significant increase in absorbance was observed for CSL16d-Pen until the pH value was greater than 9, at which point unfolding of the apo peptide contributes to the signal (see Figure S4 in the Supporting Information). This behavior is not consistent with Cd bound to three thiolate groups (either CdS3 or CdS3O). However, it would be [*] Dr. A. F. A. Peacock, Prof. V. L. Pecoraro Department of Chemistry, University of Michigan Ann Arbor, MI 48109 (USA) Fax: (+1)734-936-7628 E-mail: [email protected]