Detection of the swings of the lever arm of a myosin motor by fluorescence resonance energy transfer of green and blue fluorescent proteins.
The "lever-arm" model of a myosin motor predicts that the lever-arm domain in the myosin head tilts and swings against the catalytic domain during ATP hydrolysis, resulting in force generation. To investigate if this "swing" of the lever arm really occurs during the hydrolysis of ATP, we employed fluorescence resonance energy transfer (FRET) between two fluorescent proteins [green (GFP) and blue (BFP)] fused to the N and C termini of the Dictyostelium myosin-motor domain. FRET measurements showed that the C-terminal BFP in the fusion protein first swings against the N-terminal GFP at the isomerization step of the ATP hydrolysis cycle and then swings back at the phosphate-release step. Because the C-terminal BFP mimics the motion of the lever arm, the result indicates that the lever arm swings at the specific steps of the ATP hydrolysis cycle, i.e., at the isomerization and phosphate-release steps. The latter swing may correspond to the power stroke of myosin, while the former may be related to the recovery stroke.
Suzuki
Y
Y
Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Komaba 3-8-1, Meguro, Tokyo, 153-8902, Japan. ysuzuki@bio.c.u-tokyo.ac.jp
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001703:Biophysics / Q000379:methods*; D004023:Dictyostelium; D004735:Energy Transfer; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D008956:Models, Chemical; D008958:Models, Molecular; D020409:Molecular Motor Proteins / Q000502:physiology*; D009068:Movement / Q000502:physiology*; D009218:Myosins / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000502:physiology; D013050:Spectrometry, Fluorescence
D008164:Luminescent Proteins; D020409:Molecular Motor Proteins; D011993:Recombinant Fusion Proteins; C409504:blue fluorescent protein, Aequorea victoria; D049452:Green Fluorescent Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D009218:Myosins