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Journal of Biochemistry Advance Access published online on September 8, 2006
Journal of Biochemistry, doi:10.1093/jb/mvj188
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© 2006 The Japanese Biochemical Society.
Received July 5, 2006
Accepted September 6, 2006

Regular Paper

Probing Dynamics and Conformational Change of the GroEL-GroES Complex by 13C NMR Spectroscopy

Noritaka Nishida 1, Fumihiro Motojima 2, Mayu Idota 3, Hiroshi Fujikawa 4, Masasuke Yoshida 2, Ichio Shimada 4, and Koichi Kato 3 *

1 Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan; Graduate School of Pharmaceutical Sciences, Nagoya City University, Mizuho-ku, Nagoya 467-8603, Japan
2 The Chemical Research Laboratory, Tokyo Institute of Technology, Midori-ku, Yokohama 226-8503, Japan
3 Graduate School of Pharmaceutical Sciences, Nagoya City University, Mizuho-ku, Nagoya 467-8603, Japan
4 Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan

* To whom correspondence should be addressed.
Koichi Kato, E-mail: kkato{at}phar.nagoya-cu.ac.jp


  Abstract

Bacterial chaperonin GroEL with a molecular mass of 800 kDa was studied by 13C NMR spectroscopy. Carbonyl carbons of GroEL were labeled with 13C 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 13C 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 the trans ring 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 13C 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.

Keywords: amino acid selective labeling; 13C NMR; conformational fluctuation; GroEL; GroES.
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