Peptide Sequence and Solvent as Levers to Control Disulfide Connectivity in Multiple Cysteine Containing Venom Toxins

Judicious choice of solvent, temperature, and strategic mutations along a peptide backbone can minimize formation of non-native disulfide bond isoforms in chemical synthesis of multiple cysteine containing venom toxins. By exploiting these controls, one can drive the population distribution in favor of a particular isoform. Some chosen ionic liquids (ILs), like 1-ethyl-3-methyl-imidazolium acetate, [Im₂₁][OAc], have proven efficient in favoring the native globular isoform in some conotoxins. To comprehend such a preference, we report an explicit solvent replica exchange molecular dynamics (REMD) study of two conotoxins, AuIB and GI, solvated in either neat water or ∼50% (v/v) mixture of water-[Im₂₁][OAc]. Our simulations indicate that compared to neat water, the probability of obtaining native globular isoform of AuIB significantly increases in a water-IL mixture at 305 K. Strikingly, and aligned with experimental observations, peptide GI does not favor the native connectivity in the water-IL mixture. In presence of IL, strong solvent mediated fluctuations of the GI backbone are observed in our simulations. Uneven ion accumulation along the backbone owing to strong H-bonding interactions of some GI residues with IL ions, especially the anion OAc^-, restricts conformational freedom of the peptide. Estimation of backbone entropy and Helmholtz free energy corroborates the lack of conformational freedom in GI as compared to AuIB, especially in the presence of IL. In line with prior experiments, simulations of GI mutants indicate that one could possibly force a given pair of Cys residues to come closer by strategically mutating GI residues with glycine and/or alanine, resulting in the breakage/formation of helix-like motifs.