Probing dynamics and conformational change of the GroEL-GroES complex by 13C NMR spectroscopy
Probing dynamics and conformational change of the GroEL-GroES complex by 13C NMR spectroscopy
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DOI：
10.1093/jb/mvj188
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发表时间：
2006-10-01
影响因子：
2.7
通讯作者：
Kato, Koichi
中科院分区：
文献类型：
--
作者：
Nishida, Noritaka;Motojima, Fumihiro;Kato, Koichi
利用C-13核磁共振光谱对分子质量为800 kDa的细菌伴侣蛋白GroEL进行了研究。为了减少在光谱中观察到的信号数量，以氨基酸特异性的方式用C-13标记GroEL的羰基碳。结合选择性标记和定点诱变使我们能够建立来自GroEL的C-13共振的序列特异性分配。adp结合诱导Tyr478在赤道域和His401在中间域发生化学位移，而Tyr203在顶端域几乎不发生化学位移。在ADP存在下，Tyr478与共伴侣蛋白GroES形成络合物时，表现出来自不对称GroEL-GroES络合物的顺式环和反式环的两个峰。与GroEL复合物中GroEL共振的谱宽比较，发现拓宽与GroEL的大小不成比例，表明存在可能与伴侣活性有关的构象波动。基于这些结果，我们得出结论，结合C-13核磁共振观察和选择性标记和定点诱变可以用于探测目前核磁共振方法无法达到的超大分子的构象变化和动力学。
Bacterial chaperonin GroEL with a molecular mass of 800 kDa was studied by C-13 NMR spectroscopy. Carbonyl carbons of GroEL were labeled with C-13 in an amino acid specific manner in order to reduce the number of signals to be observed in the spectrum. Combination of selective labeling and site-directed mutagenesis enabled us to establish the sequence specific assignment of the C-13 resonances from GroEL. ADP-binding induced a chemical shift change of Tyr478 in the equatorial domain and His401 in the intermediate domain, but little of Tyr203 in the apical domain. Upon complex formation with co-chaperonin GroES in the presence of ADP, Tyr478 exhibits two peaks that would originate from the cis and trans rings of the asymmetric GroEL-GroES complex. Comparison between the line width of the GroEL resonances and those from GroES in complex with GroEL revealed broadening disproportionate to the size of GroEL, implying the existence of conformational fluctuations which may be pertinent to the chaperone activity. Based on these results, we concluded that C-13 NMR observation in combination with selective labeling and site-directed mutagenesis can be utilized for probing the conformational change and dynamics of the extremely large molecules that are inaccessible with current NMR methods.