Abstract
Cysteine sulfinic acid (Cys-SO2–) is a protein post-translational modification that is formed reversibly under oxidative conditions. A short, encodable peptide was developed whose metal binding and terbium luminescence are dependent on cysteine (Cys) oxidation to the sulfinic acid. The protein design is based on the modification of a key metal-binding aspartate (Asp) in a canonical EF-Hand motif (DKDADGWISPAEAK) to Cys. In this design, Cys in the thiol oxidation state does not mimic the native Asp, and thus the peptide binds terbium(III) (Tb3+) poorly and exhibits weak terbium luminescence (fluorescence). In contrast, when Cys is oxidized to the Cys sulfinic acid oxoform, the Cys sulfinate effectively mimics Asp, resulting in a significant increase in terbium affinity and luminescence. Asp residues at positions 1, 3, and 5 of the EF-Hand motif were examined as potential sites for Cys oxidation-responsive metal binding. The peptide with Cys at residue 1 exhibited the highest Tb3+ affinity in both oxidation states. The peptide with Cys at residue 3 exhibited a 4.2-fold distinction in affinity between the oxidation states. Most significantly, the peptide with Cys at residue 5 had only modest Tb3+ affinity as the Cys thiol, but exhibited a 30-fold increase in Tb3+ affinity and an 18-fold increase in Tb3+ luminescence on Cys oxidation to the sulfinic acid. This peptide (Ac-DKDACGWISPAEAK-NH2) exhibited selective Tb3+ binding via Cys-SO2– over the thiol, S-glutathionyl, S-nitrosyl, and sulfonic acid oxoforms, indicating substantially greater Lewis basicity of the sulfinate than the sulfonate. NMR spectroscopy and quantum homology modeling indicated that the designed peptide binds metal with an overall geometry similar to that of an EF-Hand motif, with the Cys sulfinate effectively replacing Asp as a metal-binding ligand. This peptide was applied to detect Cys oxidation to the sulfinic acid by fluorescence spectroscopy, suggesting its broader application in understanding Cys sulfinic acid biology.
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