Search

Cysteine sulfinic acid (Cys-SO 2 - ) 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) (Tb 3+ ) 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 Tb 3+ 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 Tb 3+ affinity as the Cys thiol, but exhibited a 30-fold increase in Tb 3+ affinity and an 18-fold increase in Tb 3+ luminescence on Cys oxidation to the sulfinic acid. This peptide (Ac-DKDACGWISPAEAK-NH 2 ) exhibited selective Tb 3+ binding via Cys-SO 2 - 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., Competing Interests: Declaration of competing interest There are no conflicts to declare., (Copyright © 2020. Published by Elsevier Inc.)

Cysteine sulfinic acid (Cys-SO 2 - ) is a non-enzymatic oxidative post-translational modification (PTM) that has been identified in hundreds of proteins. However, the effects of cysteine sulfination are in most cases poorly understood. Cys-SO 2 - is structurally distinctive, with long sulfur-carbon and sulfur-oxygen bonds, and with tetrahedral geometry around sulfur due to its lone pair. Cys-SO 2 - thus has a unique range of potential interactions with the protein backbone which could facilitate protein structural changes. Herein, the structural effects of cysteine oxidation to the sulfinic acid were investigated in model peptides and folded proteins using NMR spectroscopy, circular dichroism, bioinformatics, and computational studies. In the PDB, Cys-SO 2 - shows a greater preference for α-helix than Cys. In addition, Cys-SO 2 - is more commonly found in structures with φ > 0, including in multiple types of β-turn. Sulfinate oxygens engage in hydrogen bonds with adjacent (i or i + 1) amide hydrogens. Over half of sulfinates have at least one hydrogen bond with an adjacent amide, and several structures have hydrogen bonds with both adjacent amides. Alternately, sulfur or either oxygen can act as an electron donor for n→π* interactions with the backbone carbonyl of the same residue, as indicated by frequent S⋯CO or O⋯CO distances below the sums of their van der Waals radii in protein structures. In peptides, Cys-SO 2 - favored α-helical structure at the N-terminus, consistent with helix dipole effects and backbone hydrogen bonds with the sulfinate promoting α-helix. Cys-SO 2 - has only modestly greater polyproline II helix propensity than Cys-SH, likely due to competition from multiple side chain-backbone interactions. Cys-SO 2 - stabilizes the i+1 position of a β-turn relative to Cys-SH. Within proteins, the range of side chain-main chain interactions available to Cys-SO 2 - compared to Cys-SH provides a basis for potential changes in protein structure and function due to cysteine oxidation to the sulfinic acid., (Copyright © 2020 Elsevier Inc. All rights reserved.)