19F NMR studies of solvent exposure and peptide binding to an SH3 domain
19F NMR studies of solvent exposure and peptide binding to an SH3 domain
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DOI：
10.1016/j.bbagen.2006.10.017
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发表时间：
2007-02-01
影响因子：
3
通讯作者：
Prosser, R. Scott
中科院分区：
文献类型：
--
作者：
Evanics, Ferenc;Kitevski, Julianne L.;Prosser, R. Scott
用F-19 NMR研究了Fyn酪氨酸激酶SH 3结构域的拓扑结构，包括游离蛋白和与结合肽形成的复合物。将偏氟化酪氨酸生物合成地掺入SH 3结构域的G48 M突变体的5个残基中的每一个中（即，除了C-末端多组氨酸标签的接头区中的单个残基之外的残基8、10、49和54）。观察到不同的19 F NMR共振，随后在分别引入单个苯丙氨酸突变后进行分配。F-19 NMR化学位移依赖于蛋白质浓度高于0.6 mM，提示通过酪氨酸侧链附近的结合位点的二聚化。Fyn SH 3的F-19 NMR谱也作为小肽（2-羟基烟碱-NH）-Arg-Ala-Leu-Pro-Pro-Leu-Pro-二氨基丙酸-NH 2的浓度的函数获得，已知其与SH 3结构域的典型聚脯氨酸11（PPII）螺旋结合位点相互作用。基于Tyr 8、Tyr 49和Tyr 54的19 F化学位移，作为肽浓度的函数，获得18 +/- 14 μ M的平衡解离常数。对线宽的分析表明，在假定96%的FynSH 3蛋白被结合的肽浓度下，与肽-蛋白双位点交换相关的平均交换速率k（ex）为5200 +/- 600 s（-1）。溶剂暴露的氟标签的程度进行了研究，溶剂同位素位移和顺磁效应从溶解氧的组合。在不存在结合肽的情况下，除了C-末端标签上的Tyr之外，Tyr 54、Tyr 49、Tyr 10和Tyr 8似乎在间氟位置处完全暴露于溶剂。Tyr 54和在一定程度上Tyr 10在肽结合状态下受到保护而不受溶剂的影响，这与SH 3-结构域肽复合物的已知结构数据一致。这些结果显示了F-19-间氟酪氨酸与顺磁性造影剂联合探测蛋白质-蛋白质相互作用的潜在效用。(c)2006爱思唯尔B.V版权所有，保留所有权利。
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.