期刊
BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS
卷 1770, 期 2, 页码 221-230出版社
ELSEVIER
DOI: 10.1016/j.bbagen.2006.10.017
关键词
fluorine NMR; Fyn SH3; solvent exposure; oxygen; paramagnetic effect
F-19 NMR was used to study topological features of the SH3 domain of Fyn tyrosine kinase for both the free protein and a complex formed with a binding peptide. Metafluormated tyrosine was biosynthetically incorporated into each of 5 residues of the G48M mutant of the SH3 domain (i.e. residues 8, 10, 49 and 54 in addition to a single residue in the linker region to the C-terminal polyhistidine tag). Distinct 19F NMR resonances were observed and subsequently assigned after separately introducing single phenylalanine mutations. F-19 NMR chemical shifts were dependent on protein concentration above 0.6 mM, suggestive of dimerization via the binding site in the vicinity of the tyrosine side chains. F-19 NMR spectra of Fyn SH3 were also obtained as a function of concentration of a small peptide (2-hydroxynicotinic-NH)-Arg-Ala-Leu-Pro-Pro-Leu-Pro-diaminopropionic acid-NH2, known to interact with the canonical polyproline 11 (PPII) helix binding site of the SH3 domain. Based on the 19F chemical shifts of Tyr8, Tyr49, and Tyr54, as a function of peptide concentration, an equilibrium dissociation constant of 18 +/- 14 mu M was obtained. Analysis of the line widths suggested an average exchange rate, k(ex), associated with the peptide-protein two-site exchange, of 5200 +/- 600 s(-1) at a peptide concentration where 96% of the FynSH3 protein was assumed to be bound. The extent of solvent exposure of the fluorine labels was studied by a combination of solvent isotope shifts and paramagnetic effects from dissolved oxygen. Tyr54, Tyr49, Tyr10, and Tyr8, in addition to the Tyr on the C-terminal tag, appear to be fully exposed to the solvent at the metafluoro position in the absence of binding peptide. Tyr54 and, to some extent, Tyr10 become protected from the solvent in the peptide bound state, consistent with known structural data on SH3-domain peptide complexes. These results show the potential utility of F-19-metafluorotyrosine to probe protein-protein interactions in conjunction with paramagnetic contrast agents. (c) 2006 Elsevier B.V All right, reserved.
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