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Fig. 1.
Location of site-directed mutagenesis in the crystal structure of kinesin motor domain. ADP and mutational sites Cyproheptadine hydrochloride represented in the space-filling model. The structure was developed using the molecular graphic program Swiss-PdbViewer Ver.3.9b1 using the coordinate data (human ubiquitous kinesin: 1BG2) from the protein data bank database.
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Materials and methods
To measure the MT-activated ATPase activity of each mutant kinesin [16] and [18], 0.1 μM kinesin was incubated with each MT (0–10 μM) and 1 mM ATP in 50 mM imidazole–HCl (pH 6.7), 10 mM NaCl, 3 mM MgCl2, 1 mM EGTA, and 1 mM βME at 25 °C.
EPR spectroscopy. EPR measurements were performed with a Bruker ELEXSYS E500 spectrometer as described previously [16], [19], [20] and [21]. The experimental spectra were resolved into fast and slow components by spectral subtraction, as described previously [21]. Assuming cilia the experimental spectra contain only two single components corresponding to the fast and slow motion of the spin label, the fraction of one spectrum subtracted from the other required to obtain a single-component spectrum was determined. The single-component spectrum thus obtained was subtracted from the experimental spectrum to produce another single-component spectrum.