An NMR method for the determination of protein binding interfaces using TEMPOL-induced chemical shift perturbations.
An NMR method for the determination of protein binding interfaces using TEMPOL-induced chemical shift perturbations.
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DOI：
10.1016/j.bbagen.2009.06.001
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发表时间：
2009-10
期刊：
影响因子：
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通讯作者：
J. Moriya;M. Sakakura;Yuji Tokunaga;R. Prosser;I. Shimada
中科院分区：
文献类型：
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作者：
J. Moriya;M. Sakakura;Yuji Tokunaga;R. Prosser;I. Shimada
蛋白质-蛋白质界面的确定对于理解蛋白质的功能和指导化合物的设计至关重要。自由扩散的4-羟基-2，2，6，6-四甲基哌啶-1-氧自由基（TEMPOL）诱导的~（13）C NMR顺磁位移是利用NMR光谱识别蛋白质-蛋白质界面的一个很有前途的方法，因为TEMPOL影响了目标蛋白质溶剂可及核的~（13）C NMR化学位移，方法通过记录13 C-NMR谱，测定了TEMPO诱导的13 C NMR顺磁位移，并与13 C-NMR谱进行了比较，结果表明，TEMPO诱导的13 C-NMR谱的顺磁位移与13 C-NMR谱的顺磁位移基本一致。泛素在游离状态和与酵母泛素水解酶1（YUH 1）的复合状态下的13 C TOCSY光谱。泛素与YUH 1复合后，与蛋白质结合界面相关的13 C NMR顺磁位移减少0.05ppm或更多。结论TEMPOL诱导的13 C化学位移微扰法是一种测定蛋白质-蛋白质界面的有效方法，具有样品制备简单、数据分析简便、适用范围广等优点。
BACKGROUNDThe determination of protein–protein interfaces is of crucial importance to understand protein function and to guide the design of compounds. To identify protein–protein interface by NMR spectroscopy,13C NMR paramagnetic shifts induced by freely diffusing 4-hydroxy-2, 2, 6, 6-tetramethyl-piperidine-1-oxyl (TEMPOL) are promising, because TEMPOL affects distinct13C NMR chemical shifts of the solvent accessible nuclei belonging to proteins of interest, while13C nuclei within the interior of the proteins may be distinguished by a lack of such shifts.METHODWe measured the13C NMR paramagnetic shifts induced by TEMPOL by recording13C–13C TOCSY spectra for ubiquitin in the free state and the complex state with yeast ubiquitin hydrolase1 (YUH1).RESULTSUpon complexation of ubiquitin with YUH1,13C NMR paramagnetic shifts associated with the protein binding interface were reduced by 0.05 ppm or more. The identified interfacial atoms agreed with the prior X-ray crystallographic data.CONCLUSIONSThe TEMPOL-induced13C chemical shift perturbation is useful to determine precise protein–protein interfaces.GENERAL SIGNIFICANCEThe present method is a useful method to determine protein–protein interface by NMR, because it has advantages in easy sample preparations, simple data analyses, and wide applicabilities.