Mechanical and chemical properties of cysteine-modified kinesin molecules.
To probe the structural changes within kinesin molecules, we made the mutants of motor domains of two-headed kinesin (4-411 aa) in which either all the five cysteines or all except Cys45 were mutated. A residual cysteine (Cys45) of the kinesin mutant was labeled with an environment-sensitive fluorescent probe, acrylodan. ATPase activity, mechanical properties, and fluorescence intensity of the mutants were measured. Upon acrylodan-labeled kinesin binding to microtubules in the presence of 1 mM AMPPNP, the peak intensity was enhanced by 3.4-fold, indicating the structural change of the kinesin head by the binding. Substitution of cysteines decreased both the maximum microtubule-activated ATPase and the sliding velocity to the same extent. However, the maximum force and the step size were not affected; the force produced by a single molecule was 6-6.5 pN, and a step size due to the hydrolysis of one ATP molecule by kinesin molecules was about 10 nm for all kinesins. This step size was close to a unitary step size of 8 nm. Thus, the mechanical events of kinesin are tightly coupled with the chemical events.
Iwatani
S
S
Department of Biophysical Engineering, Osaka University, Japan.
Iwane
A H
AH
Higuchi
H
H
Ishii
Y
Y
Yanagida
T
T
D015081:2-Naphthylamine / Q000031:analogs & derivatives / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D019878:Carboxyl and Carbamoyl Transferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002417:Cattle; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004330:Drosophila; D005456:Fluorescent Dyes / Q000378:metabolism; D006868:Hydrolysis; D016547:Kinesins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D007834:Lasers; D008958:Models, Molecular; D020409:Molecular Motor Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; D020409:Molecular Motor Proteins; D011993:Recombinant Fusion Proteins; C038518:acrylodan; D015081:2-Naphthylamine; D019878:Carboxyl and Carbamoyl Transferases; C021580:Methylmalonyl-CoA carboxytransferase; D000251:Adenosine Triphosphatases; D016547:Kinesins; D003545:Cysteine