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Assessing the Influence of Mutation on GTPase Transition States by using X-ray Crystallography, 19F NMR, and DFT Approaches
Angewandte Chemie International Edition ( IF 16.1 ) Pub Date : 2017-05-24 08:50:50 , DOI: 10.1002/anie.201703074
Yi Jin 1, 2 , Robert W. Molt 2, 3, 4 , Erika Pellegrini 5 , Matthew J. Cliff 6 , Matthew W. Bowler 5, 7 , Nigel G. J. Richards 2 , G. Michael Blackburn 1 , Jonathan P. Waltho 1, 6
Affiliation  

We report X-ray crystallographic and 19F NMR studies of the G-protein RhoA complexed with MgF3, GDP, and RhoGAP, which has the mutation Arg85′Ala. When combined with DFT calculations, these data permit the identification of changes in transition state (TS) properties. The X-ray data show how Tyr34 maintains solvent exclusion and the core H-bond network in the active site by relocating to replace the missing Arg85′ sidechain. The 19F NMR data show deshielding effects that indicate the main function of Arg85′ is electronic polarization of the transferring phosphoryl group, primarily mediated by H-bonding to O3G and thence to PG. DFT calculations identify electron-density redistribution and pinpoint why the TS for guanosine 5′-triphosphate (GTP) hydrolysis is higher in energy when RhoA is complexed with RhoGAPArg85′Ala relative to wild-type (WT) RhoGAP. This study demonstrates that 19F NMR measurements, in combination with X-ray crystallography and DFT calculations, can reliably dissect the response of small GTPases to site-specific modifications.

中文翻译:

使用X射线晶体学,19F NMR和DFT方法评估突变对GTPase过渡态的影响

我们报告的X射线晶体学和19个G蛋白RhoA的络合氟化镁的F NMR研究3 -,GDP和RhoGAP,它具有突变Arg85'Ala。与DFT计算结合使用时,这些数据可以识别过渡状态(TS)属性的变化。X射线数据显示Tyr34如何通过重新定位来替换缺失的Arg85'侧链,从而在活性位点保持溶剂排除和核心H键网络。的19个F NMR数据显示去屏蔽指示Arg85的主要功能效应“是所述转印磷酰基的电子极化,主要由氢键介导至O 3G并从那里至P ģ。DFT计算确定了电子密度的重新分布,并指出了当RhoA与RhoGAP Arg85'Ala相比于野生型(WT)RhoGAP时,鸟苷5'-三磷酸(GTP)水解的TS在能量上更高的原因。这项研究表明19 F NMR测量与X射线晶体学和DFT计算相结合,可以可靠地剖析小GTP酶对位点特异性修饰的响应。
更新日期:2017-05-25
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