Nishida Noritaka, Motojima Fumihiro, Idota Mayu, Fujikawa Hiroshi, Yoshida Masasuke, Shimada Ichio, Kato Koichi
Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033 Japan.
J Biochem. 2006 Oct;140(4):591-8. doi: 10.1093/jb/mvj188. Epub 2006 Sep 8.
Bacterial chaperonin GroEL with a molecular mass of 800 kDa was studied by (13)C NMR spectroscopy. Carbonyl carbons of GroEL were labeled with (13)C 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 (13)C 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 (13)C 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.
利用（13）C核磁共振波谱对分子量为800 kDa的细菌伴侣蛋白GroEL进行了研究。为了减少光谱中待观测信号的数量，以氨基酸特异性方式用（13）C标记GroEL的羰基碳。选择性标记与定点诱变相结合，使我们能够确定GroEL的（13）C共振的序列特异性归属。ADP结合诱导赤道结构域中的Tyr478和中间结构域中的His401发生化学位移变化，但顶端结构域中的Tyr203几乎没有变化。在ADP存在下与共伴侣蛋白GroES形成复合物时，Tyr478出现两个峰，这可能源于不对称GroEL - GroES复合物的顺式和反式环。GroEL共振的线宽与与GroEL形成复合物的GroES的线宽之间的比较显示，加宽与GroEL的大小不成比例，这意味着存在可能与伴侣活性相关的构象波动。基于这些结果，我们得出结论，（13）C核磁共振观测与选择性标记和定点诱变相结合，可用于探测目前核磁共振方法无法触及的超大分子的构象变化和动力学。
