Design, synthesis, and characterization of 4-ester CI2, a model for backbone hydrogen bonding in protein α-helices

The total synthesis of proteins enables unnatural groups to be incorporated into proteins to understand the molecular basis of protein stability and function. Chymotrypsin inhibitor 2 (CI2), is a small 64 residue protein consisting of an alpha -helix sandwiched by four beta -strands. To directly evaluate the role of backbone hydrogen bonding in alpha -helices, an array of four amide bonds that span the length of the alpha -helix have been replaced with ester bonds. Residues 13, 16, 19, and 22 have been substituted with alpha -hydroxy acids using solid-phase synthesis, and the peptides were assembled by conformationally assisted ligation. The resulting 4-ester CI2 is a functional protease inhibitor that is destabilizeh by 2.93 kcal/mol compared to the all-amide protein. This study demonstrates that the formation of multiple consecutive hydrogen bonds is not required for the folding of protein alpha -helices.