Structural kinetics of myosin by transient time-resolved FRET.

Abstract

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.

• Authors: Yuri E Nesmelov, Roman V Agafonov, Igor V Negrashov, Sarah E Blakely, Margaret A Titus, David D Thomas
• Journal: Proceedings of the National Academy of Sciences of the United States of America (Proc Natl Acad Sci U S A) Vol. 108 Issue 5 Pg. 1891-6 (Feb 01 2011) ISSN: 1091-6490 [Electronic] United States
• PMID: 21245357 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)

Chemical References

• Adenosine Triphosphate
• Myosins

Topics

• Adenosine Triphosphate (chemistry)
• Dictyostelium (chemistry)
• Fluorescence Resonance Energy Transfer (methods)
• Kinetics
• Myosins (chemistry)
• Protein Conformation
• Spectrometry, Fluorescence