Ryan L Stafford | PubMed Health

PubMed2003

EF-Tu binding peptides identified, dissected, and affinity optimized by phage display.

The highly abundant GTP binding protein elongation factor Tu (EF-Tu) fulfills multiple roles in bacterial protein biosynthesis. Phage-displayed peptides with high affinity for EF-Tu were selected from a library of approximately 4.7 x 10(11) different peptides. The lack of sequence homology among the identified EF-Tu ligands demonstrates promiscuous peptide binding by EF-Tu. Homolog shotgun scanning of an EF-Tu ligand was used to dissect peptide molecular recognition by EF-Tu. All homolog shotgun scanning selectants bound to EF-Tu with higher affinity than the starting ligand. Thus, homolog shotgun scanning can simultaneously optimize binding affinity and rapidly provide detailed structure activity relationships for multiple side chains of a polypeptide ligand. The reported peptide ligands do not compete for binding to EF-Tu with various antibiotic EF-Tu inhibitors, and could identify an EF-Tu peptide binding site distinct from the antibiotic inhibitory sites.

Amino Acid Sequence↗

PubMed2002

Dissecting the streptavidin-biotin interaction by phage-displayed shotgun scanning.

Shotgun scanning the streptavidin-biotin interaction identifies long-range hydrophobic interactions that contribute to one of the strongest naturally occurring noncovalent protein-ligand interactions. The femtomolar dissociation constant for this interaction makes it a useful model system to dissect the forces that govern high-affinity molecular recognition between proteins and small molecules. Shotgun scanning combines the diversity and in vitro binding selection of phage-displayed libraries with a binomial mutagenesis strategy. Libraries consist of proteins with the residues in multiple positions mutated to give a 1:1 ratio of alanine:wild type. Here, we use shotgun scanning to determine the functional contribution of the 38 C-terminal residues of streptavidin to the high-affinity interaction with biotin. The library pools were subjected to three rounds of selection for functional streptavidin variants that bind biotin and statistical analysis was used to assess side-chain contributions to biotin binding. The results demonstrate the utility of shotgun scanning for the dissection of receptor-small-molecule interactions. While shotgun scanning results were largely consistent with previous single-point, site-directed mutagenesis studies for residues in direct contact with biotin, residues distant from the biotin binding site have not previously been explored. Key streptavidin residues identified by shotgun scanning as contributors to the interaction with biotin include those with side chains that fill the beta barrel, residues at the tetramer interface, and second-sphere residues, which are reinforced by long-distance propagation of hydrophobic interactions.

Binding Sites↗