Synthetic approaches to elucidate roles of disulfide bridges in peptides and proteins

Abstract

The pairing of cysteine residues to form disulfide bridges represents the only way that nature establishes covalent crosslinks bringing together in three dimensions portions of linear polypeptide chains that are apart in the linear sequence. As a consequence, conformations of peptides and proteins can be “locked” and both their stabilities and biological activities are affected. Disulfide bridges can also be harnessed in creative ways for de novo design studies. Finally, an active research question is to understand the roles of disulfide bridges in the folding process; the answer may be different for protein sequences able to readily access their final stable packed conformations versus peptide molecules that are generally quite conformationally flexible until covalent crosslinks are introduced. In order to develop a better understanding of the relative importance of disulfides, our laboratory continues to develop methods for the reliable assembly of cystine-containing peptides and small proteins, and for the construction of the required bridges [1]. We also consider a variety of analogous structures, including those in which disulfides are replaced by trisulfides, in which disulfides have been intentional mispaired, and in which paired half-cysteines have been replaced by paired acid isosteres. These approaches are applied to a range of target molecules, including oxytocin and deaminooxytocin (9 residues, disulfide bridge between residues 1 and 6), somatostatin (14 residues, disulfide bridge between residues 3 and 14), SI (13 residues, disulfide bridges between residues 2 and 7; 3 and 13), apamin (18 residues, disulfide bridges between residues 1 and 11; 3 and 15), and bovine pancreatic trypsin inhibitor (BPTI, 58 residues, disulfide bridges between residues 5 and 55; 14 and 38; and 30 and 51). This article surveys our most recent progress both on synthetic aspects and on conformational/biological consequences, some of which is detailed further in the Proceedings of this Symposium [2]. We report new cysteine protecting groups and racemization-free anchoring and coupling procedures, a reusable polymeric reagent to mediate intramolecular disulfide formation, and studies which compare solution and onresin regioselective schemes for disulfide bridge formation.