Mechanical and chemical properties of cysteine-modified kinesin molecules

To probe the structural changes within kinesin molecules, we made the mutan ts of motor domains of two-headed kinesin (4-411 aa) in which either all th e five cysteines or all except Cys45 were mutated. A residual cysteine (Cys 45) of the kinesin mutant was labeled with an environment-sensitive fluores cent probe, acrylodan. ATPase activity, mechanical properties, and fluoresc ence 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 m icrotubule-activated ATPase and the sliding velocity to the same extent. Ho wever, the maximum force and the step size were not affected; the force pro duced by a single molecule was 6-6.5 pN, and a step size due to the hydroly sis of one ATP molecule by kinesin molecules was about 10 nm for all kinesi ns. This step size was close to a unitary step size of 8 nm. Thus, the mech anical events of kinesin are tightly coupled with the chemical events.