Use of peptide libraries to define protein binding specificity
Diagnostic Techniques and Procedures
Protein kinases find their downstream targets by a series of selection procedures based on the structure of the kinase and its target. To address the specificity of domains involved in protein kinase/substrate interactions we developed a novel "oriented peptide library" technique. This technique has now been used to define optimal peptide motifs for binding to SH 2 domains, SH3 domains, LIM domains. PDZ domains, proline isomerases and 14-3-3 proteins. These results, combined with structural information obtained from NMR and x-ray crystallography, have provided a basis for explaining how individual members of families of domains recognize distinct proteins. We also developed a library of oriented peptide substrates for kinases and were able to determine the optimal nonapeptide substrates for more than 30 protein-tyrosine kinases and protein-Ser/Thr kinases. These results can be rationalized based on crystal structures of some of the kinases. The structural models proposed are being investigated by mutational studies. This approach not only provides a structural basis for explaining how protein kinases find their targets hut also allows predictions of new targets based on primary sequences ot proteins.