Exploring Nature's Inventory: Investigating the Role of Amide to Thioamide Substitution on Protein Stability

Abstract

The peptide backbone holds a protein together and plays a crucial role in guiding its three-dimentional structure. The tertiary structure of proteins regulates several biological processes. Therefore, peptide bond modification has gained significant attention to influence protein folding, stability, and functions. However, it is a challenging task to mimic the peptide (amide) bond due to its unique properties, such as planar structure, hydrogen bond donor, and acceptor properties. Thioamide, a single O to S substitution in an amide bond, is the closest isostere and has shown promising results on small peptides. The recent discovery of thioamide in natural protein, methyl-coenzyme M reductase (MCR), raises an important fundamental question of its role in protein conformation and stability. However, the synthesis of thio modified peptides/proteins is challenging. Therefore, we first focused on the synthetic procedure for the synthesis of thioamidated peptides and proteins. We have shown the compatibility of our synthetic method in incorporating the thionated derivative of all the 20 naturally occurring amino acids onto a growing peptide chain. We also report the use of a 2% DBU + 5% piperazine cocktail for fast Fmoc-deprotection that allowed us to synthesize thioamidated Pin1 WW domain and GB1 directly on a solid support. Next, we demonstrated the role of a single n→π* interaction on protein stability by engineering n→π* interaction at the β-turn. Our experimental results at the i+2 residue of type-II’ β-turn in GB1 variants suggest that amino acid side-chain identity and the rotamer conformation can modulate the strength of an n→π* interaction. The altered rotamer conformation as a result of local structural changes within a protein can amplify/weaken an n→π* interaction affecting the backbone torsion angles (phi, psi), and thereby influencing its stability. Further, we amplified the strength of n→π* interaction by replacing i+1 donor carbonyl with thiocarbonyl and validated that the n→π* interaction can indeed influence the structural stability of proteins. We also highlighted the potential impact of a single atom substitution in stabilizing the β-sheet protein. It broadens the scope of this backbone mutation approach in designing/stabilizing the protein scaffold.