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Volumn 283, Issue 5405, 1999, Pages 1152-1157

A processive single-headed motor: Kinesin superfamily protein KIF1A

Author keywords

[No Author keywords available]

Indexed keywords

KINESIN;

EID: 0033582814     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.283.5405.1152     Document Type: Article
Times cited : (374)

References (52)
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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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    • 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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    • -1, so that we can almost neglect the effect of photobleaching in the motility assays.
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    • note
    • 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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    • note
    • 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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    • 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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    • 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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    • note
    • MT9ATPase) was >200 nM, indicating that the affinity of C351 for MTs was reduced by a factor of 10.
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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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    • 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.
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    • 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.
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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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    • 2.
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    • note
    • 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.


* 이 정보는 Elsevier사의 SCOPUS DB에서 KISTI가 분석하여 추출한 것입니다.