shown to involve an interaction between the rt loop of hck sh3 and residues in nef outside of its prototypic polyproline type 11 ppii helix containing sh3 ligand region such distinctive interactions are thought to provide specificity and affinity for other sh3 ligand protein complexes as well in this invention we constructed and successfully displayed on the surface of m13 bacteriophage particles a complex library of sh3 domains which are derived from hck but carry a random hexapeptide substitution in their rt loops termed rrt sh3 by this strategy we identified individual rrt sh3 domains that can bind to nef with affinities higher than k d 10 nm some of these high affinity rrt sh3 domains resembled hck sh3 in that they bound much less well to a nef variant containing an engineered f90r mutation that interferes with docking of the native hck rt loop in addition we could also select rrt sh3 domains with an opposite specificity which were dependent on the arg 90 residue for strong binding and had a 100 fold lower affinity for unmodified nef these results emphasize the importance of the rt loop in sh3 ligand selection and suggest a general strategy for creating sh3 domains with desired binding properties to examine if it would be possible to present functional sh3 domains on the surface of bacteriophages we constructed phagemid containing hck sh3 and by using a m13ko7 helper virus produced recombinant phages expressing it fused to the piii coat protein when these phages were incubated in six well plates coated with purified gst nef gst nef pa1 an sh3 binding deficient p72a p75a mutant of nef or plain gst protein.