Design of Antibodies Specific for Unfolded Proteins

Protein unfolding leads to aggregation and loss of function. No antibodies have been reported that recognize generic conformational epitopes within unfolded proteins despite the importance of detecting protein unfolding at extremely low concentrations and in the presence of background (folded) proteins. We posit that antibodies specific for solvent-exposed aromatic residues (which are normally solvent-shielded within folded proteins) will selectively recognize unfolded conformations of diverse proteins. Based on this hypothesis, we have engineered the complementarity determining regions of small antibodies with aromatic repeat sequences to enable them to bind to unfolded proteins via intermolecular pi-stacking interactions. We find that such antibodies recognize diverse proteins in their unfolded state and bind only to solvent-exposed aromatic residues. In contrast, the same aromatic repeat peptides (apart from the antibody) bind weakly to unfolded proteins, revealing that constraint of aromatic peptides within antibody loops significantly increases their binding affinity. We will discuss how these novel antibodies can be used to monitor protein unfolding in a highly sensitive and site-specific manner.