Divergent Protein Synthesis of Bowman–Birk Protease Inhibitors, their Hydrodynamic Behavior and Co-crystallization with α-Chymotrypsin

Abstract

A divergent protein synthesis strategy was executed to effectively synthesize Bowman–Birk protease inhibitor (BBI) analogues using native chemical ligation of peptide hydrazides. Grafting selected residues from a potent trypsin inhibitor, sunflower trypsin inhibitor-1, onto the α-chymotrypsin-binding loop of BBI, resulted in a fourfold improvement of α-chymotrypsin inhibition. The crystal structure of a synthetic BBI analogue co-crystallized with α-chymotrypsin confirmed the correct protein fold and showed a similar overall structure to unmodified BBI in complex with α-chymotrypsin. Dynamic light scattering showed that C-terminal truncation of BBI led to increased self-association.