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Attempts to Visualize Conformational Changes in a Single Protein Molecule During Function

Published online by Cambridge University Press:  02 July 2020

Kazuhiko Kinosita Jr. ,
Ryohei Yasuda ,
Ichiro Sase  and
Hidetake Miyata
Kazuhiko Kinosita Jr.
Affiliation:
Department of Physics, Faculty of Science and Technology, Keio University, Hiyoshi 3-14-1, Kohoku-ku, Yokohama, 223, Japan
Ryohei Yasuda
Affiliation:
Department of Physics, Faculty of Science and Technology, Keio University, Hiyoshi 3-14-1, Kohoku-ku, Yokohama, 223, Japan
Ichiro Sase
Affiliation:
Present address:Microscopes Development Department, Yokohama Plant, Nikon Corporation, Nagaodai-machi 471, Sakae-ku, Yokohama244, Japan
Hidetake Miyata
Affiliation:
Present address:Physics Department, Graduate School of Science, Tohoku University, Aoba, Aramaki, Aoba-ku, Sendai980-77, Japan
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Extract

The operation of protein molecular machines is essentially stochastic. One cannot predict exactly when an ion channel opens, or when a molecular motor makes a step. As such, working molecular machines cannot be synchronized with each other in a rigorous sense. To understand the mechanism of a protein machine, therefore, one has to watch conformational changes of individual molecules while they are at work. To this end, we have been trying to develop two approaches based on optical microscopy. One is to attach a small tag, a fluorescent probe, to an appropriate site on the protein molecule of interest. Polarization of the probe fluorescence, for example, will reveal the orientation of the fluorophore, and thus of the portion of the protein molecule to which the fluorophore is attached. A conformational change of the protein will be detected as the reorientation of the fluorophore. A prerequisite is the ability to image a single fluorophore in an aqueous environment, which we have achieved on an epifluorescence microscope by reducing its background luminescence by two orders of magnitude

Type
Light Microscopy: Recent Advances
Information
Microscopy and Microanalysis , Volume 3 , Issue S2: Proceedings: Microscopy & Microanalysis '97, Microscopy Society of America 55th Annual Meeting, Microbeam Analysis Society 31st Annual Meeting, Histochemical Society 48th Annual Meeting, Cleveland, Ohio, August 10-14, 1997 , August 1997 , pp. 799 - 800
Copyright
Copyright © Microscopy Society of America 1997

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References

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This work is the result of collaboration with Dr. S. Ishiwata's group at Waseda University and with Dr. M. Yoshida's group at Tokyo Institute of Technology. We thank Mr. H. Itoh and Mr. M. Hosoda (Hamamatsu Photonics) for image processing. Supported by Grants-in-Aid from Ministry of Education, Science, Sports and Culture of Japan and a Keio University Special Grant-in-Aid for Innovative Collaborative Research Projects, and by CREST (Core Research for Evolutional Science and Technology) of Japan Science and Technology Corporation.Google Scholar