Synthesis of Scyllatoxin-Based BH3 Domain Mimetics with Diverse Patterns of Native Disulfide Bonds

This article outlines the design and development of scyllatoxin (ScTx)-based BH3 domain mimetics with diverse patterns of native disulfide bonds. More specifically, this method summarizes the total chemical synthesis of ScTx-based peptides that contain zero, one, two, or three disulfide linkages, including techniques to generate variants with any combination of native disulfides. Each peptide reported herein is generated on solid-phase support using microwave-assisted coupling procedures, and all reaction parameters related to the peptide synthesis are described in detail. The various disulfide patterns of the ScTx-based constructs are established during peptide synthesis and are ultimately verified by mass analysis of trypsin-digested fragments. The BH3 domain mimetics developed herein were generated by transposing residues from the helical BH3 domain of the pro-apoptotic BCL2 protein Bax to the α-helix of wild-type ScTx. Interestingly, we found that the relative binding affinities of ScTx-Bax peptides for the anti-apoptotic BCL2 protein Bcl-2 (proper) were heavily influenced by the number and position of disulfide linkages within the ScTx-Bax sequence. As a consequence, we were able to utilize ScTx-Bax BH3 domain mimetics with varied patterns of disulfide bonds to survey how structural rigidity within the helical Bax BH3 domain affects binding to promiscuous anti-apoptotic BCL2 proteins. More broadly, the ability to generate ScTx-based molecules that contain any combination of native disulfide bonds expands the utility of such constructs as tools to study the molecular nature of protein-protein interactions. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Synthesis and characterization of ScTx-based Bax BH3 domain mimetics Basic Protocol 2: Oxidation of ScTx-Bax BH3 domain mimetics containing one, two, or three disulfide linkages Support Protocol: Mapping of disulfide linkages in oxidized ScTx-Bax BH3 domain mimetics.