The highly abundant GTP binding protein elongation factor Tu (EF-Tu) fulfills multiple roles in bacterial protein biosynthesis. EF-Tu also binds other ligands, including four structurally distinct families of antibiotics. The lack of sequence homology among the identified EF-Tu ligands demonstate promiscuous peptide binding by EF-Tu.
A peptide library with a diversity of ~ 4.7X1011 different peptides was constructed containing both linear and disulfide-constrained peptides. Phage-displayed peptides with high affinity for EF-Tu were selected from the library. Phage display was also used to rapidly assess the contribution of specific side chains to EF-Tu binding through combinatorial libraries of alanine and homolog substitutions termed "shotgun scanning".
Homolog shotgunning provided detailed structure activity relationships for multiple side chains of polypeptide ligands. Additional binding assays showed that phage-displayed peptide ligands do not compete for binding to EF-Tu with various antibiotic EF-Tu inhibitors, and these ligands could identify an EF-Tu peptide binding site distinct from the antibiotic inhibitory sites.
This phage-display peptide library is about 10,000X more diverse than commercially available libraries. It may be used to screen for EF-Tu ligands that may lead to potential new antibacterial therapies. It may also be used to screen for new non-EF-Tu targets that bind to the ligands in this diverse library.