The WW domain is an approximately 38 residue peptide-binding
motif that binds 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 β-sheet. Improved binding
to the hydrophobic peptide, GTPPPPYTVG (WW1), was selected
in the presence of three different concentrations of proteinase
K to simultaneously drive selection for improved stability
as well as high-affinity binding. While some of the selected
binders show cooperative unfolding transitions, others
show noncooperative thermal unfolding curves. Two novel
WW consensus sequences have been identified, which bind
to the xPPxY motif with higher affinity than the wild-type
hYAP65 WW domain. These WW domain sequences are not precedented
in any natural WW domain sequence. Thus, there appear to
be a large number of motifs capable of recognizing the
target peptide sequence, only a subset of which appear
to be used in natural proteins.