Peptide Inhibitor Assay for Allocating Functionally Important Accessible Sites Throughout a Protein Chain:...

Functionally important amino acid sequences in proteins are often located at multiple sites. Three-dimensional structural analysis and site-directed mutagenesis may be performed to allocate functional sites for understanding structure‒function relationships and for developing novel inhibitory drugs. However, such methods are too demanding to comprehensively cover potential functional sites throughout a protein chain. Here, a peptide inhibitor assay (PIA) was devised to allocate functionally important accessible sites in proteins. This simple method presumes that protein‒ligand interactions, intramolecular interactions, and dimerization interactions can be partially inhibited by high concentrations of competitive "endogenous" peptides of the protein of interest. Focusing on the restriction endonuclease EcoRI as a model protein system, many endogenous peptides (6mer-14mer) were synthesized, covering the entire EcoRI protein chain. Some of them were highly inhibitory, but interestingly, the nine most effective peptides were located outside the active sites, with the exception of one. Relatively long peptides with aromatic residues (F, H, W, and Y) corresponding to secondary structures were generally effective. Because synthetic peptides are flexible enough to change length and amino acid residues, this method may be useful for quickly and comprehensively understanding structure‒function relationships and developing novel drugs or epitopes for neutralizing antibodies.