SOLUTION STUDIES OF THE SH2 DOMAIN FROM THE FYN TYROSINE KINASE - SECONDARY STRUCTURE, BACKBONE DYNAMICS AND PROTEIN ASSOCIATION

The SH2 domain from Fyn tyrosine kinase, corresponding to residues 155 -270 of the human enzyme, was expressed as a GST-fusion protein in a p GEX-E. coli system. After thrombin cleavage and removal of GST, the pr otein was studied by heteronuclear NMR. Two different phosphotyrosyl-p eptides were synthesized and added to the SH2 domain. One peptide corr esponded to the regulatory C-terminal tail region of Fyn. Sequence-spe cific assignment of NMR spectra was achieved using a combination of H- 1-N-15-correlated 2D HSQC, N-15-edited 3D TOCSY-HMQC, and N-15-edited 3D NOESY-HMQC spectra. tra. By analysis of the ex-proton chemical shif ts and NOE intensities, the positions of secondary structural elements were determined and found to correspond closely to that seen in the c rystal structure of the, homologous, Src-SH2 domain. To investigate th e internal dynamics of the protein backbone, T-1 and T-2 relaxation pa rameters were measured on the free protein, as well as on both peptide complexes. Analytical ultracentrifugation and dynamic light scatterin g were employed to measure the effect of concentration and peptide-bin ding on self-association. The results suggest that, at NMR-sample conc entrations, the free protein is present in at least dimeric form. Phos phopeptide binding and lower concentration significantly, but not comp letely, shift the equilibrium towards monomers. The possible role of t his protein association in the regulation of the Src-family tyrosine k inases is discussed.