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6 tag for purification and a reactive cysteine residue for labeling. These constructs were expressed with the pET expression system (Novagen) in Escherichia coli. Bacterially expressed proteins were purified by immobilized metal affinity chromatography and labeled with Alexa-594 maleimide (Molecular Probes) at a fluorochrome/protein ratio of 0.7 to 0.9.
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19
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0345282438
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note
-
There is a lack of consensus with regard to the terminology of the domains of kinesin, or KIFs. Here, we refer to the region from β1 to α6 as the "catalytic core" region, the following region before α7 as the "linker," and the first coiled-coil α7 (the essential domain for the dimerization) as the "neck."
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T. Funatsu, Y. Harada, M. Tokunaga, K. Saito, T. Yanagida, Nature 374, 555 (1995); M. Tokunaga, K. Kitamura, K. Saito, A. H. Iwane, T. Yanagida, Biochem. Biophys. Res. Commun. 235, 47 (1997). Motor proteins were labeled with Alexa 594 C5-maleimide (Molecular Probes), and MTs were labeled with Bodipy-FL (Molecular Probes). Alexa 594 was selected because of its high photostability, large Stokes shift, high extinction coefficient, and high quantum yield. Motor proteins were filtered through ultrafiltration membranes just before use to remove aggregates. The filtrate was immediately diluted with motility buffer [50 mM imidazole, 5 mM Mg-acetate, 1 mM EGTA, 50 mM K-acetate, 10 mM dithiothreitol, 0.1% Triton X-100, casein (1 mg/ml), 2 mM ATP, and 10 μM paclitaxel] containing an oxygen scavenger system [1% glucose, glucose oxidase (0.05 mg/ml), and catalase (0.1 mg/ml)] [Y. Harada, K. Sakurada, T. Aoki, D. D. Thomas, T. Yanagida, J. Mol. Biol. 216, 49 (1990)]. The temperature of the specimen stage was controlled at 26° ± 1°C by the air conditioner of the clean room. Epifluorescent images were observed under an Olympus BX microscope with a PlanApo 100X/1.40 objective lens and filter sets suited to Bodipy and Alexa (Olympus). Any part not necessary for the epifluorescent imaging was removed. An excitation beam passed through the sample was projected to the wall of the room so as not to cause reflection back into the light path of the emitted fluorescence. These improvements effectively reduced the background noise level (Fig. 1C) below the fluorescence signals of single Alexa molecules. The fluorescence images were projected to a Gen IV image-intensified cooled charge-coupled device camera (V/1CCD, Princeton Instruments) and electronically amplified with an image processor (Argus-10, Hamamatsu Photonics, Japan). The images were integrated for eight frames to improve the signal/noise ratio. Thus, the spatial and temporal resolution of this system is 50 nm (= pixel size) and 8/30 s, respectively. The resultant images were further analyzed on a Macintosh computer using the public-domain program NIH Image (available at http://rsb.info.nih.gov/nih-image/) with a custom macro program for semi-automatic quantification of fluorescent intensity and displacement.
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0344419508
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note
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-1, so that we can almost neglect the effect of photobleaching in the motility assays.
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27
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0344419509
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note
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Previous reports on the single-motor assay of conventional kinesin (4) use mean run-length of the motor as the index of the mechanical processivity. However, this parameter is sensitively affected by the occasional backward movement. Therefore, it is not suitable for C351 as shown in Fig. 2C.
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31
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0027137481
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0344850996
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note
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diss for dimerization is >1 μM, whereas the assays were performed at <10 pM) and the fluorescent intensity distribution of C351 (Fig. 1C). However, there still remains a possibility that the fluorescent labeling might have affected the dimerization and that non-fluorescent C351 might preferentially associate with fluorescently labeled molecules.
-
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36
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0345282436
-
-
note
-
MT-activated ATPase activity was assayed with the EnzChek phosphate assay kit (Molecular Probes). MT-activated ADP release was measured by the method of Hackney (8, 9, 26). The reactions were performed in the motility buffer without the oxygen scavenger system, and all assays were performed at 26° ± 1°C.
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37
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0344850995
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-
note
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The kinetic parameters of Alexa-labeled C351 were also measured to assess the effects of fluorescent labeling. The parameters of Alexa-labeled C351 were not different from those of unlabeled C351, which effectively excludes the possibility that Alexa labeling changed the behavior of C351.
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38
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0344419505
-
-
note
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MT9ATPase) was >200 nM, indicating that the affinity of C351 for MTs was reduced by a factor of 10.
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39
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0028985886
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S. P. Gilbert, M. R. Webb, M. Brune, K. A. Johnson, Nature 373, 671 (1995); Y. Z. Ma and E. W. Taylor, Biochemistry 34, 13242 (1995).
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S. P. Gilbert, M. R. Webb, M. Brune, K. A. Johnson, Nature 373, 671 (1995); Y. Z. Ma and E. W. Taylor, Biochemistry 34, 13242 (1995).
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45
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0345282433
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note
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MT(ATPase) value was more than five times that of C351. In the single-motor assay, this mutant showed no binding to MTs, hence no processive movement was observed.
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46
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0031471243
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F. Kozielski et al., Cell 91, 985 (1997).
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0345714037
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note
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1/2]. To encompass this displacement by a 6-nm motor, N should be >20, which gives a mean mechanical step size of 0.065 nm, smaller than the size of the atom. Other models, such as a loose-coupling model with constant mechanical step size and fluctuating coupling ratio (N), also give subatomic step size.
-
-
-
-
48
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0345713353
-
-
note
-
From thermodynamics theory, the observed kinetic or mechanical parameters collectively give the difference between C351 and K3B1 in the depth of the potential as ∼4kT, where T is the absolute temperature and k is the Boltzmann constant. The extra lysines in the K-loop would make this difference in the binding energy.
-
-
-
-
49
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0031042321
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p ≊ 1 to 2 nm. Thus, this model can quantitatively explain the observed biased Brownian movement of C351 with parameters of reasonable values.
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Amos, L.A.1
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50
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0345714015
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note
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2.
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52
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0345714036
-
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note
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Supported by a Center of Excellence grant-in-aid from the Ministry of Education, Science, Sports, and Culture of Japan (N.H.). We thank K. Abe for his technical assistance with the microscopy system, H. Sato and H. Fukuda for their secretarial assistance, M. Sugaya for her technical assistance, and M. Kikuchi, M. Kikkawa, and other members of our lab for discussions and suggestions.
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