title: Isolation of monobodies that bind specifically to the SH3 domain of the Fyn tyrosine protein kinase.
abstract: Fyn is a nonreceptor protein tyrosine kinase that belongs to a highly conserved kinase family, Src family kinases. Fyn plays an important role in inflammatory processes and neuronal functions. To generate a synthetic affinity reagent that can be used to probe Fyn, a phage-display library of fibronectin type III monobodies was affinity selected with the Src Homology 3 (SH3) domain of Fyn and three binders were isolated. One of the three binders, G9, is specific in binding to the SH3 domain of Fyn, but not to the other members of the Src family (i.e. Blk, Fgr, Hck, Lck, Lyn, Src and Yes), even though they share 51-81% amino acid identity. The other two bind principally to the Fyn SH3 domain, with some cross-reactivity to the Yes SH3 domain. The G9 binder has a dissociation constant of 166±6nM, as measured by isothermal titration calorimetry, and binds only to the Fyn SH3 domain out of 150 human SH3 domains examined in an array. Interestingly, although the G9 monobody lacks proline in its randomized BC and FG loops, it binds at the same site on the SH3 domain as proline-rich ligands, as revealed by competition assays. The G9 monobody, identified in this study, may be used as a highly selective probe for detecting and purifying cellular Fyn kinase.
authors.affiliation: Department of Biological Sciences, University of Illinois at Chicago, 845 W. Taylor St., 3240 SES - MC 066, Chicago, IL 60607-7060, USA.
authors.forename: Renhua
authors.initials: R
authors.lastname: Huang
authors.forename: Pete
authors.initials: P
authors.lastname: Fang
authors.forename: Brian K
authors.initials: BK
authors.lastname: Kay
mesh_terms: D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology*; D001665:Binding Sites; D040761:Biomimetic Materials / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D002151:Calorimetry; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D019151:Peptide Library; D011485:Protein Binding; D025941:Protein Interaction Mapping; D051076:Proto-Oncogene Proteins c-fyn / Q000737:chemistry* / Q000378:metabolism*; D016415:Sequence Alignment; D018909:src Homology Domains
chemical_list: D000906:Antibodies; D019151:Peptide Library; D051076:Proto-Oncogene Proteins c-fyn