| Structural kinetics of myosin by transient time-resolved FRET. | |
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MedLine Citation:
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PMID: 21245357 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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For many proteins, especially for molecular motors and other enzymes, the functional mechanisms remain unsolved due to a gap between static structural data and kinetics. We have filled this gap by detecting structure and kinetics simultaneously. This structural kinetics experiment is made possible by a new technique, (TR)(2)FRET (transient time-resolved FRET), which resolves protein structural states on the submillisecond timescale during the transient phase of a biochemical reaction. (TR)(2)FRET is accomplished with a fluorescence instrument that uses a pulsed laser and direct waveform recording to acquire an accurate subnanosecond time-resolved fluorescence decay every 0.1 ms after stopped flow. To apply this method to myosin, we labeled the force-generating region site specifically with two probes, mixed rapidly with ATP to initiate the recovery stroke, and measured the interprobe distance by (TR)(2)FRET with high resolution in both space and time. We found that the relay helix bends during the recovery stroke, most of which occurs before ATP is hydrolyzed, and two structural states (relay helix straight and bent) are resolved in each nucleotide-bound biochemical state. Thus the structural transition of the force-generating region of myosin is only loosely coupled to the ATPase reaction, with conformational selection driving the motor mechanism. |
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Authors:
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Yuri E Nesmelov; Roman V Agafonov; Igor V Negrashov; Sarah E Blakely; Margaret A Titus; David D Thomas |
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Publication Detail:
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Type: Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't Date: 2011-01-18 |
Journal Detail:
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Title: Proceedings of the National Academy of Sciences of the United States of America Volume: 108 ISSN: 1091-6490 ISO Abbreviation: Proc. Natl. Acad. Sci. U.S.A. Publication Date: 2011 Feb |
Date Detail:
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Created Date: 2011-02-02 Completed Date: 2011-03-23 Revised Date: 2012-02-13 |
Medline Journal Info:
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Nlm Unique ID: 7505876 Medline TA: Proc Natl Acad Sci U S A Country: United States |
Other Details:
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Languages: eng Pagination: 1891-6 Citation Subset: IM |
Affiliation:
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Department of Biochemistry, University of Minnesota Medical School, Minneapolis, MN 55455, USA. yuri.nesmelov@uncc.edu |
Export Citation:
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| MeSH Terms | |
Descriptor/Qualifier:
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Adenosine Triphosphate
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chemistry Dictyostelium / chemistry Fluorescence Resonance Energy Transfer / methods* Kinetics Myosins / chemistry* Protein Conformation Spectrometry, Fluorescence |
| Grant Support | |
ID/Acronym/Agency:
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AG26160/AG/NIA NIH HHS; AR32961/AR/NIAMS NIH HHS; AR53562/AR/NIAMS NIH HHS; AR59621/AR/NIAMS NIH HHS; GM27906/GM/NIGMS NIH HHS; R01 AR032961-29/AR/NIAMS NIH HHS; R37 AG026160-07/AG/NIA NIH HHS |
| Chemical | |
Reg. No./Substance:
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56-65-5/Adenosine Triphosphate; EC 3.6.4.1/Myosins |
| Comments/Corrections | |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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