2004 Fiscal Year Final Research Report Summary
Elucidation of the input-output characteristics of F_1-motor
| Project/Area Number |
14380313
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Biophysics
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| Research Institution | Tokyo Institute of Technology |
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Principal Investigator |
EIRO Muneyuki Tokyo Institute of Technology, Chemical Resources Laboratory, research associate, 資源化学研究所, 助手 (80219865)
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| Project Period (FY) |
2002 – 2004
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| Keywords | ATP synthase / free energy / energy transduction / rotary motor / work / heat / ratchet mode / fluctuation |
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| Research Abstract |
Biological motor proteins convert free energy of ATP hydrolysis to mechanical work, thus playing essential roles in life. But the fundamental question how they work at different free energy of ATP hydrolysis has been unanswered. This question becomes serious when we ask how stepping motion of a single motor molecule in a medium reflects the concentrations of ATP. ADP and inorganic phosphate in their individual actions. The F_1 portion of ATP synthase, also called as F_1-ATPase, is a rotary molecular motor in which central γ subunit rotates against α_3β_3 cylinder hydrolyzing ATP. Its structure is highly stable. The rotary action of this motor is processive and generates high torque. These features are ideal to explore the relationship between input free energy and output mechanical work except that this motor protein is severely inhibited by ADP. Here we overcame this problem by introducing several mutations while keeping high enzymatic activity. With a probe of beads attached, steppin
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g rotation against viscous load was examined at a wide range of free energy by changing ADP concentration. The results showed that the work of individual step motion was not affected by the free energy of ATP hydrolysis but their frequency depended on the free energy of ATP showing a property of a molecular machine operating in an environment dominated by Brownian motion.
In addition, we have analyzed rotations of a mutant F_1 subcomplex (E190D) which hydrolyzes ATP slowly and rotations of the wild-type F_1 employing a slowly hydrolysable substrate ATPγS (adenosine-5'-(γ-thio)-triphosphate). In both cases, interim dwells at 80° position were extended. We concluded that that cleavage of ATP takes place during the interim dwell.
Furthermore, we could observe rotation under an optical microscope, while watching which of the three sites bound and released a fluorescent ATP analog. If we assume that the analog mimics authentic ATP, the following scheme emerges : (i)in the ATP-waiting state, one site, dictated by the orientation of γ, is empty whereas the other two bind a nucleotide ; (ii)ATP binding to the empty site drives 〜80° rotation of γ ; (iii)this triggers a reaction(s), hydrolysis and/or phosphate release but not ADP release, in the site that bound ATP one step ago ; (iv)completion of that reaction induces further 〜40° rotation.
Thus, we could elucidate important features of the energetics and kinetics of the F1-motor. Less
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[Journal Article] Chemo-mechanical coupling in F_1-ATPase revealed by simultaneous observation of nucleotide kinetics and rotation.2004
Author(s)
Nishizaka, T., Oiwa, K., Noji, H., Kimura, S., Muneyuki, E., Yoshida, M., Kinosita, K.Jr.
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Journal Title
Nature Structural & Molecular Biology 11
Pages: 142-148
Description
「研究成果報告書概要(和文)」より
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[Journal Article] Catalysis and rotation of F_1 motor, cleavage of ATP at the catalytic site occurs in 1ms before 40°substep rotation2003
Author(s)
Shimabukuro, K., Yasuda, R., Muneyuki, E., Hara, K.Y., Kinosita, K.Jr.Yoshida, M.
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Journal Title
Proc.Natl.Acad.Sci.USA 100
Pages: 14731-14736
Description
「研究成果報告書概要(和文)」より
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[Journal Article] Catalysis and rotation of F_1 motor ; cleavage of ATP at the catalytic site occurs in 1 ms before 40° substep rotation2003
Author(s)
Shimabukuro, K., Yasuda, R., Muneyuki.E., Hara, K.Y., Kinosita, K.Jr., Yoshida, M.
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Journal Title
Proc.Natl.Acad.Sci.USA 100
Pages: 14731-14736
Description
「研究成果報告書概要(欧文)」より
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