Redox-Responsive Protein Design: Design of a Small Protein Motif Dependent on Glutathionylation

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 for probing 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. Nuclear magnetic resonance 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.