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Journal of Biochemistry Advance Access originally published online on September 8, 2006
Journal of Biochemistry 2006 140(4):591-598; doi:10.1093/jb/mvj188
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© 2006 The Japanese Biochemical Society.

ARTICLE

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

Noritaka Nishida1,2, Fumihiro Motojima3, Mayu Idota2, Hiroshi Fujikawa1, Masasuke Yoshida3, Ichio Shimada1 and Koichi Kato2,*

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

* To whom correspondence should be addressed. Tel: +81-52-836-3447, Fax: +81-52-836-3447, E-mail: kkato{at}phar.nagoya-cu.ac.jp

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 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 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.


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