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Kinetic Analysis of Conformational Changes of GroEL Based on the Fluorescence of Tyrosine 506

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Abstract

The conformational changes of GroEL during the ATPase cycle in the presence of GroES were studied by measuring the fluorescence intensity time course of intrinsic tyrosine Y506, which is located near the nucleotide-binding site. A GroEL solution containing GroES was mixed with an ATP solution to initiate the reaction cycle. The tyrosine fluorescence intensity relative to that without the nucleotide reached 112% within the dead time of the apparatus (>15 s−1) and further increased to 123% at 0.57 s−1 followed by a decrease to 102% at 0.32 s−1. An initial conformational change and a second intermediate state were expected to occur in ATP-bound GroEL because similar changes were observed for the ATPase-deficient D398A mutant. The conformational change to the third intermediate state corresponded to a process during or after ATP hydrolysis because D398A had no decreasing phase. The second intermediate state before ATP hydrolysis was characterized for the first time.

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Abbreviations

AEX:

A mutant of GroEL (C138S/C458S/C519S/D83C/K327C)

AEXred :

Reduced AEX

AEXox :

Oxidized AEX

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Acknowledgments

This work was supported in part by the Mitsubishi Foundation (T.F.) and by Grants-in-Aid for Scientific Research on Priority Areas (H.T., M.Y.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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Authors and Affiliations

  1. Major in Integrated Bioscience and Biomedical Engineering, Graduate School of Science and Engineering, Waseda University, Shinjuku-ku, Tokyo, 169-8555, Japan

    Kazuhiko Hosono

  2. Laboratory of Bio-Analytical Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan

    Taro Ueno, Tamotsu Zako & Takashi Funatsu

  3. Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa-shi, Chiba, 277-8562, Japan

    Hideki Taguchi

  4. Precursory Research for Embryonic Science and Technology (PRESTO), JST, Chiyoda-ku, Tokyo, 102-0075, Japan

    Hideki Taguchi

  5. Chemical Resources Laboratory, Tokyo Institute of Technology, Midori-ku, Yokohama, 226-8503, Japan

    Fumihiro Motojima & Masasuke Yoshida

  6. Center for NanoBio Integration, The University of Tokyo, Bunkyo-ku, Tokyo, 113-8656, Japan

    Takashi Funatsu

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  1. Kazuhiko Hosono
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  2. Taro Ueno
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  3. Hideki Taguchi
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  4. Fumihiro Motojima
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  5. Tamotsu Zako
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  6. Masasuke Yoshida
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  7. Takashi Funatsu
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Correspondence to Takashi Funatsu.

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Hosono, K., Ueno, T., Taguchi, H. et al. Kinetic Analysis of Conformational Changes of GroEL Based on the Fluorescence of Tyrosine 506. Protein J 27, 461–468 (2008). https://doi.org/10.1007/s10930-008-9157-9

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  • DOI: https://doi.org/10.1007/s10930-008-9157-9

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