Synthesis and conformational preferences of peptides and proteins with cysteine sulfonic acid

Abstract

Cysteine sulfonic acid (Cys-SO₃H; cysteic acid) is an oxidative post-translational modification of cysteine, resulting from further oxidation from cysteine sulfinic acid (Cys-SO₂H). Cysteine sulfonic acid is considered an irreversible post-translational modification, which serves as a biomarker of oxidative stress that has resulted in oxidative damage to proteins. Cysteine sulfonic acid is anionic, as a sulfonate (Cys-SO₃⁻; cysteate), in the ionization state that is almost exclusively present at physiological pH (pK_a ∼ −2). In order to understand protein structural changes that can occur upon oxidation to cysteine sulfonic acid, we analyzed its conformational preferences, using experimental methods, bioinformatics, and DFT-based computational analysis. Cysteine sulfonic acid was incorporated into model peptides for α-helix and polyproline II helix (PPII). Within peptides, oxidation of cysteine to the sulfonic acid proceeds rapidly and efficiently at room temperature in solution with methyltrioxorhenium (MeReO₃) and H₂O₂. Peptides containing cysteine sulfonic acid were also generated on solid phase using trityl-protected cysteine and oxidation with MeReO₃ and H₂O₂. Using methoxybenzyl (Mob)-protected cysteine, solid-phase oxidation with MeReO₃ and H₂O₂ generated the Mob sulfone precursor to Cys-SO₂⁻ within fully synthesized peptides. These two solid-phase methods allow the synthesis of peptides containing either Cys-SO₃⁻ or Cys-SO₂⁻ in a practical manner, with no solution-phase synthesis required. Cys-SO₃⁻ had low PPII propensity for PPII propagation, despite promoting a relatively compact conformation in ϕ. In contrast, in a PPII initiation model system, Cys-SO₃⁻ promoted PPII relative to neutral Cys, with PPII initiation similar to Cys thiolate but less than Cys-SO₂⁻ or Ala. In an α-helix model system, Cys-SO₃⁻ promoted α-helix near the N-terminus, due to favorable helix dipole interactions and favorable α-helix capping via a sulfonate-amide side chain–main chain hydrogen bond. Across all peptides, the sulfonate side chain was significantly less ordered than that of the sulfinate. Analysis of Cys-SO₃⁻ in the PDB revealed a very strong propensity for local (i/i or i/i + 1) side chain–main chain sulfonate–amide hydrogen bonds for Cys-SO₃⁻, with >80% of Cys-SO₃⁻ residues exhibiting these interactions. DFT calculations conducted to explore these conformational preferences indicated that side chain–main chain hydrogen bonds of the sulfonate with the intraresidue amide and/or with the i + 1 amide were favorable. However, hydrogen bonds to water or to amides, as well as interactions with oxophilic metals, were weaker for the sulfonate than the sulfinate, due to lower charge density on the oxygens in the sulfonate.