Arginine residues are broadly employed for specific biomolecular recognition, including in protein–protein, protein–DNA, and protein–RNA interactions. Arginine recognition commonly exploits the potential for bidentate electrostatic and hydrogen-bonding interactions. However, in arginine residues, the guanidinium functional group is located at the terminus of a flexible hydrocarbon side chain, which lacks the functionality to contribute to specific arginine-mediated recognition and may entropically disfavor binding. In order to enhance the potential for specificity and affinity in arginine-mediated molecular recognition, we have developed an approach to the synthesis of peptides that incorporates an α-guanidino acid as a novel arginine mimetic. α-Guanidino acids, derived from α-amino acids, with guanidinylation of the amino group, were incorporated stereospecifically into peptides on solid phase via coupling of an Fmoc amino acid to diaminopropionic acid (Dap), Fmoc deprotection, guanidinylation of the amine on solid phase, and deprotection, generating a peptide containing an α-functionalized arginine mimetic. This approach was examined by incorporating arginine mimetics into ligands for the Src, Grb, and Crk SH3 domains at the site of the key recognition arginine. Protein binding was examined for peptides containing guanidino acids derived from Gly, L-Val, L-Phe, L-Trp, D-Val, D-Phe, and D-Trp. We demonstrate that paralogue specificity and target site affinity may be modulated with the use of α-guanidino acid-derived arginine mimetics, generating peptides that exhibit enhanced Src specificity by selection against Grb and peptides that reverse the specificity of the native peptide ligand, with enhancements in Src target specificity of up to 15-fold (1.6 kcal mol⁻¹).

Lanthanides have interesting chemical properties; these include luminescent, magnetic, and catalytic functions. Toward the development of proteins incorporating novel functions, we have designed a new lanthanide-binding motif, lanthanide fingers. These were designed based on the Zif268 zinc finger, which exhibits a ββα structural motif. Lanthanide fingers utilize an Asp₂Glu₂ metal-coordination environment to bind lanthanides through a tetracarboxylate peptide ligand. The iterative design of a general lanthanide-binding peptide incorporated the following key elements: 1) residues with high α-helix and β-sheet propensities in the respective secondary structures; 2) an optimized big box α-helix N-cap; 3) a Schellman α-helix C-cap motif; and 4) an optional D-Pro-Ser type II’ β-turn in the β-hairpin. The peptides were characterized for lanthanide binding by circular dichroism (CD), NMR, and fluorescence spectroscopy. In all instances, stabilization of the peptide secondary structures resulted in an increase in metal affinity. The optimized protein design was a 25-residue peptide that was a general lanthanide-binding motif; this binds all lanthanides examined in a competitive aqueous environment, with a dissociation constant of 9.3 μM for binding Er³⁺. CD spectra of the peptide-lanthanide complexes are similar to those of zinc fingers and other ββα proteins. Metal binding involves residues from the N-terminal β-hairpin and the C terminal α-helical segments of the peptide. NMR data indicated that metal binding induced a global change in the peptide structure. The D-Pro-Ser type II’ β-turn motif could be replaced by Thr–Ile to generate genetically encodable lanthanide fingers. Replacement of the central Phe with Trp generated genetically encodable lanthanide fingers that exhibited terbium luminescence greater than that of an EF-hand peptide.