Measuring kinesin's first step.
A variety of models have recently emerged to explain how the molecular motor kinesin is able to maintain processive movement for over 100 steps. Although these models differ in significant features, they all predict that kinesin's catalytic domains intermittently separate from each other as the motor takes 8-nm steps along the microtubule. Furthermore, at some point in this process, one molecule of ATP is hydrolyzed per step. However, exactly when hydrolysis and product release occur in relation to this forward step have not been established. Furthermore, the rate at which this separation occurs as well as the speed of motor stepping onto and release from the microtubule have not been measured. In the absence of this information, it is difficult to critically evaluate competing models of kinesin function. We have addressed this issue by developing spectroscopic probes whose fluorescence is sensitive to motor-motor separation or microtubule binding. The kinetics of these fluorescence changes allow us to directly measure how fast kinesin steps onto and releases from the microtubule and provide insight into how processive movement is maintained by this motor.
Rosenfeld
Steven S
SS
Department of Neurology, University of Alabama at Birmingham and Neurology Service, Department of Veterans Affairs Medical Center, Birmingham, Alabama 35294, USA. stevensr@uab.edu
Xing
Jun
J
Jefferson
Geraldine M
GM
Cheung
Herbert C
HC
King
Peter H
PH
D000244:Adenosine Diphosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D016880:Anisotropy; D003545:Cysteine / Q000737:chemistry; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004735:Energy Transfer; D005456:Fluorescent Dyes / Q000494:pharmacology; D006868:Hydrolysis; D016547:Kinesins / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D008870:Microtubules / Q000378:metabolism*; D008954:Models, Biological; D008958:Models, Molecular; D009154:Mutation; D011189:Potassium Chloride / Q000494:pharmacology; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013997:Time Factors
D005456:Fluorescent Dyes; D008024:Ligands; D000244:Adenosine Diphosphate; D011189:Potassium Chloride; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D016547:Kinesins; D003545:Cysteine