Cysteine S-glutathionylation is a protein post-translational modification that promotes cellular responses to changes in oxidative conditions. The design of protein motifs that directly depend on defined changes to protein side chains provides new methods to probe diverse protein post-translational modifications. A canonical, 12-residue EF Hand motif was redesigned to be responsive to cysteine glutathionylation. The key design principle was the replacement of the metal-binding Glu12 carboxylate of an EF Hand with a motif capable of metal binding via a free carboxylate in the glutathione-conjugated peptide. In the optimized peptide (DKDADGWCG), metal binding and terbium luminescence were dependent on glutathionylation, with weaker metal binding in the presence of reduced cysteine, but increased metal affinity and a 3.5-fold increase in terbium luminescence at 544 nm when cysteine was glutathionylated. NMR spectroscopy indicated that the structure at all residues of the glutathionylated peptide changed in the presence of metal, with chemical shift changes consistent with the adoption of an EF-Hand-like structure in the metal-bound glutathionylated peptide. This small protein motif consists of canonical amino acids, and is thus genetically encodable, for its potential use as a localized tag to probe protein glutathionylation.

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 guanylation of the amino group, were incorporated stereospecifically into peptides on solid phase via coupling of an Fmoc amino acid to diaminoproprionic acid (Dap), Fmoc deprotection, guanylation of the amine on solid phase, and deprotection, generating a peptide containing an α-functionalized arginine mimetic. This approach was examined via the incorporation of 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 paralog specificity and target site affinity may be modulated via the use of α-guanidino acid-derived arginine mimetics, generating peptides that exhibit enhanced Src specificity via 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).