Controlling Disulfide Formation and Dimerization to Constrain Alternate Peptide Conformations

Disulfide bonds form covalent bonds between distal regions of peptides and proteins to dramatically impact their folding, stability, and oligomerization. Given the prevalence of disulfide bonds in many natural products, considerable effort has been invested in site-selective disulfide bond formation approaches to control folding of chemically synthesized peptides and proteins. Here we show that careful choice of thiol oxidation conditions can lead to monomeric or dimeric species from fully deprotected linear bisthiol peptides. Examination of reaction conditions indicates that the use of disulfiram (DSF) under denaturing conditions leads to the formation of intramolecular disulfide bonds while reactions with dimethyl sulfoxide (DMSO) under aqueous conditions yields disulfide-linked, antiparallel dimeric species. Intramolecular disulfide formation using DSF and denaturing conditions proved to be sequence-independent. On the other hand, the formation of antiparallel dimers is sequence-dependent and requires partial α-helical folding of the linear peptide and the presence of a hydrophobic triad to encourage peptide association. Treatment with Proteinase K indicates that both disulfide species demonstrate enhanced protease stability compared to the linear precursor. The half-life increases by a factor of 100 for the dimer and even greater for the intramolecular disulfide. Additionally, both disulfide species are fully reduced by tris(2-carboxyethyl)phosphine (TCEP) within 15 minutes to regain the original α-helical structure for targeting proteins. These results provide an approach for using disulfide bonds to control peptide folding and oligomerization to better understand how these properties influence interactions with protein and membrane targets.¹

1) Victorio, C.G. and Sawyer, N. (2023) Folding-Assisted Peptide Disulfide Formation and Dimerization. ACS Chem. Biol. 18, 1480. DOI: 10.1021/acschembio.3c00268