In the present paper, we described our attempt to characterize the rough three-dimensional features of the structural analogue of the key intermediate of myosin's cross-bridge cycle. Using quick-freeze deep-etch replica electron microscopy, we observed that actin-attached myosin during in vitro sliding was bent superficially as postulated by the conventional hypothesis, but in the opposite direction of the putative pre-power-stroke configuration, as for ADP·Vi (inorganic vanadate)-bound myosin. We searched for the conformational species with a similar appearance and found that SH1–SH2 (thiols 1 and 2)-cross-linked myosin is a good candidate. To characterize such small asymmetric structures, we employed a new pattern-recognition procedure that accommodates the metal-replicated samples. In this method, the best-matched views of the target microscopic images were selected from a comprehensive set of images simulated from known atomic co-ordinates of relevant proteins. Together with effective morphological filtering, we could define the conformational species and the view angles of the catalytic domain and the lever arm cropped from averaged images of disulfide-cross-linked myosin. Whereas the catalytic domain of the new conformer closely resembled the pPDM (N,N′-p-phenylenedimaleimide)-treated, but SH2 Lys705-cross-linked, structure (PDB code 1L2O), a minor product of the same cross-linking reaction, the lever arm projected differently. Using separately determined view angles of the catalytic domain and the lever arm, we built a model of disulfide-cross-linked myosin. Further combination with the ‘displacement-mapping’ procedure enabled us to reconstruct the global three-dimensional envelope of the unusual structure whose lever arm orientation is compatible with our reports on the actin-sliding cross-bridge structure. Assuming this conformer as the structural analogue of the transient intermediate during actin sliding, the power stroke of the lever arm might accompany the reversal of the disorganized SH1 helix.
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Research Article|
January 24 2013
Novel configuration of a myosin II transient intermediate analogue revealed by quick-freeze deep-etch replica electron microscopy Available to Purchase
Yoshitaka Kimori;
Yoshitaka Kimori
1
*Division of Biomolecular Imaging, Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
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Norio Baba;
Norio Baba
†Department of Computer Science, Faculty of Informatics, Kogakuin-University, Hachioji, Tokyo 192-0015, Japan
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Eisaku Katayama
Eisaku Katayama
2
*Division of Biomolecular Imaging, Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
2To whom correspondence should be addressed. Present address: Department of Applied Chemistry and Biotechnology, Graduate School of Engineering, Chiba University, Yayoi-cho, Inage-ku, Chiba-shi 263-8522, Japan (email [email protected]).
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Publisher: Portland Press Ltd
Received:
March 09 2012
Revision Received:
October 31 2012
Accepted:
December 05 2012
Accepted Manuscript online:
December 05 2012
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2013 Biochemical Society
2013
Biochem J (2013) 450 (1): 23–35.
Article history
Received:
March 09 2012
Revision Received:
October 31 2012
Accepted:
December 05 2012
Accepted Manuscript online:
December 05 2012
Citation
Yoshitaka Kimori, Norio Baba, Eisaku Katayama; Novel configuration of a myosin II transient intermediate analogue revealed by quick-freeze deep-etch replica electron microscopy. Biochem J 15 February 2013; 450 (1): 23–35. doi: https://doi.org/10.1042/BJ20120412
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