The Redox-Active Conopeptide Derived from the Venom Duct Transcriptome of Conus lividus Assists in the Oxidative Folding of Conotoxin.

The tetrapeptides Li504 and Li520, differing in the modification of the 4-trans-hydroxylation of proline, are novel conopeptides derived from the venom duct transcriptome of the marine cone snail Conus lividus. These predicted mature peptides are homologous to the active site motif of oxidoreductases that catalyze the oxidation, reduction, and rearrangement of disulfide bonds in peptides and proteins. The estimated reduction potential of the disulfide of Li504 and Li520 is within the range of disulfide reduction potentials of oxidoreductases, indicating that they may catalyze the oxidative folding of conotoxins. Conformational features of Li504 and Li520 include the trans configuration of the Cys1-Pro2/Hyp2 peptide bond with a type 1 turn that is similar to the active site motif of glutaredoxin that regulates the oxidation of cysteine thiols to disulfides. Li504- and Li520-assisted oxidative folding of α-conotoxin ImI confirms that Li520 improves the yield of the natively folded peptide by concomitantly decreasing the yield of the non-native disulfide isomer and thus acts as a miniature disulfide isomerase. The geometry of the Cys1-Hyp2 peptide bond of Li520 shifts between the trans and cis configurations in the disulfide form and thiol/thiolate form, which regulates the deprotonation of the N-terminal cysteine residue. Hydrogen bonding of the hydroxyl group of 4-trans-hydroxyproline with the interpeptide chain unit in the mixed disulfide form may play a vital role in shifting the geometry of the Cys1-Hyp2 peptide bond from cis to trans configuration. The Li520 conopeptide together with similar peptides derived from other species may constitute a new family of "redox-active" conopeptides that are integral components of the oxidative folding machinery of conotoxins.

Ligand-induced transition in conformations of vicinal cysteine disulfides in proteins.

Vicinal cysteine disulfides are thought to be associated with specific conformations of cysteine disulfides due to the restricted rotation of single bonds in an eight-membered cyclic disulfide loop. Conformations of vicinal cysteine disulfides are analyzed using χ1 , χ2 , χ3 , χ2 ', χ1 ' torsion angles in the crystal structures of proteins retrieved from Protein Data Bank (PDB). 85% of vicinal disulfides have (+, -)LHStaple conformation with trans configuration of the peptide bond and 9% have (-, -)RHStaple conformation with cis configured peptide bond. Conformational analysis of dipeptide Cys-Cys vicinal disulfide by density functional theory (DFT) further supported (+, -)LHStaple, (-, -)RHStaple, and (+, +)RHStaple as the preferred conformations of vicinal disulfides. Interestingly, the rare conformations of vicinal disulfides are observed in the ligand-bound forms of proteins and have higher disulfide strain energy. Conformations of vicinal disulfides in palmitoyl protein thioesterase 1, AChBP, and α7 nicotinic receptor are changed from preferred (+, -)LHStaple to rare (+, -)AntiLHHook/(+, -)AntiRHHook/(+, +)RHStaple conformation due to binding of ligands. Surprisingly, ligands are proximal to the vicinal disulfides in protein complexes that exhibited rare conformations of vicinal disulfides. The report has identified (+, -) LHStaple/(-, -) RHStaple as the hallmark conformations of vicinal disulfides and unraveled ligand-induced transition in conformations of vicinal cysteine disulfides in proteins.

Characterization of (Boc-Cys/Sec-NHMe)2 and (Boc-Cys/Sec-OMe)2 : Evidence of local conformational difference between disulfide and diselenide.

Conformations of disulfide and diselenide were compared in (Boc-Cys/Sec-NHMe)2 and (Boc-Cys/Sec-OMe)2 using X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, density functional theory (DFT), and circular dichroism (CD) spectroscopy. Conformations of disulfide/diselenide in polypeptides are defined based on the sign of side chain torsion angle χ3 (-CH2 -S/Se-S/Se-CH2 -); negative indicates left-handed and positive indicates right-handed orientation. In the crystals of (Boc-Cys-OMe)2 and (Boc-Sec-OMe)2 , the disulfide exhibits a left-handed and the diselenide a right-handed orientation. Characterization of cystine and selenocystine derivatives in solution using 1 H-NMR, natural abundant 77 Se NMR, 2D-ROESY, and chemical shift analysis coupled to DMSO titration has indicated the symmetrical nature and antiparallel orientation of Cys/Sec residues about the disulfide/diselenide bridges. Structural calculations of cystine and selenocystine derivatives using DFT further support the antiparallel orientation of Cys/Sec residues about disulfide/diselenide. The far-ultraviolet (UV) region CD spectra of cystine and selenocystine derivatives have exhibited the negative Cotton effect (CE) for disulfide and positive for diselenide confirming the difference in the conformational preference of disulfide and diselenide. In the previously reported polymorphic structure of (Boc-Sec-OMe)2 , the diselenide has right-handed orientation. In the X-ray structures of disulfide and diselenide analogues of Escherichia coli protein encoded by curli specific gene C (CgsC) retrieved from Protein Databank (PDB), disulfide has left-handed and the diselenide right-handed orientation. The current report provides the evidence for the local conformational difference between a disulfide and a diselenide group under unconstrained conditions, which may be useful for the rational replacement of disulfide by diselenide in polypeptide chains.

Peptide Cysteine Thiols Act as Photostabilizer of Avobenzone through Stabilizing the Transition State of Keto-Enol Tautomerization.

Photostabilizers have been used to impart stability to an FDA-approved chemical UV-A filter avobenzone against the UV-A radiations and sunlight. The thiol group of glutathione plays a critical role in imparting the photostabilization activity of glutathione on avobenzone. The current report aims to evaluate the photostabilization activity of multiple thiols containing cysteine peptides on avobenzone. Cysteine-tripeptide and cysteine-pentapeptide were chemically synthesized and characterized using mass spectrometry. Synthetic peptides were assessed for their photostabilization activity on the enolic-form of the avobenzone under natural sunlight using UV spectroscopy in both protic and aprotic solvents. Unlike glutathione, which has pronounced activity in protic solvents, cysteine-pentapeptide exhibits similar photoprotection activity in both protic and aprotic solvents. Computational calculations using DFT suggest that peptide cysteine thiols may assist in the reversal of the photoketonization process of avobenzone thereby exhibiting the photoprotection activity to the enolic-form of avobenzone. Peptide cysteine thiols lower the activation energy barrier of keto-to-enol tautomerization of avobenzone by 30 kcal mol-1 by assisting the proton shuttle through a six-membered transition state. The current report emphasizes the applications of peptide thiols in cosmetics and may help in the development of peptides as aesthetic medicines.

Conformational change due to replacement of disulfide with selenosulfide and diselenide in dipeptide vicinal cysteine loop.

Replacement of disulfide bridges with diselenide bridges is increasingly common to improve the stability, foldability, and structural refinement of the cysteine-rich polypeptides. Even though the global structural features are similar due to the replacement of disulfide with diselenide, the local conformational differences have been reported in a few polypeptides. The current report has used the constrained vicinal cysteine disulfide as the model to access the influence of the replacement of disulfides with diselenide on the local conformations. Using the density functional theory (DFT), structures of dipeptide vicinal loops are optimized by systematically replacing sulfur with selenium. Conformations of the disulfide/selenosulfide/diselenide were identified using the side-chain torsional angle χ1, χ2, χ3, χ2', χ1' and mapped to one of the possible 32 conformations of the cysteine disulfide. Further, the influence of the change of configuration of Cα-atom of cysteine/selenocysteine from 'R' to 'S' configuration and peptide bond from cis to trans has also been accessed on the conformations of dipeptide vicinal loops. The results indicate that diselenide/selenosulfide explores additional conformational space apart from accommodating the conformations observed in the vicinal disulfide which is more amplified in the heterochiral system. Differences have been observed at the internal coordinates of the optimized structures of dipeptide vicinal disulfide, selenosulfide, and diselenide. The change in free energy (ΔG), spin density (Δs(r)), and electron density (Δρ(r)) was also calculated due to the replacement of disulfide with selenosulfide/diselenide. Conformational analysis of disulfides and that of the replaced diselenides in the crystal structures of the proteins retrieved from PDB have also indicated the retention as well as differences in the local conformations. The tendency of the diselenide loop to explore the additional conformational space may prompt for the local conformational differences in the corresponding disulfide to diselenide replaced polypeptides.