Evolution of binding affinity in a WW domain probed by phage display

The WW domain is an approximately 38 residue peptide-binding motif that bin ds a variety of sequences, including the consensus sequence xPPxY. We have displayed hYAP65 WW on the surface of M13 phage and randomized one-third of its three-stranded antiparallel beta -sheet. Improved binding to the hydro phobic peptide, GTPPPPYTVG (WW1), was selected in the presence of three dif ferent concentrations of proteinase K to simultaneously drive selection for improved stability as well as high-affinity binding. While some of the sel ected binders show cooperative unfolding transitions, others show noncooper ative thermal unfolding curves. Two novel WW consensus sequences have been identified, which bind to the xPPxY motif with higher affinity than the wil d-type hYAP65 WW domain. These WW domain sequences are not precedented in a ny natural WW domain sequence. Thus, there appear to be a large number of m otifs capable of recognizing the: target peptide sequence, only a subset of which appear to be used in natural proteins.