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What kinesin does at roadblocks: the coordination mechanism for molecular walking.

Crevel, Isabelle M?TC, Nyitrai, Miklos, Alonso, Maria C., Weiss, Stefan, Geeves, Michael A., Cross, Robert A. (2004) What kinesin does at roadblocks: the coordination mechanism for molecular walking. EMBO Journal, 23 (1). pp. 23-32. ISSN 0261-4189. E-ISSN 1460-2075. (doi:10.1038/sj.emboj.7600042) (Access to this publication is currently restricted. You may be able to access a copy if URLs are provided) (KAR id:26)

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http://dx.doi.org/10.1038/sj.emboj.7600042

Abstract

Competing models for the coordination of processive stepping in kinesin can be tested by introducing a roadblock to prevent lead head attachment. We used T93N, an irreversibly binding mutant monomer, as a roadblock, and measured the rates of nucleotide-induced detachment of kinesin monomers or dimers with and without the T93N roadblock using microflash photolysis combined with stopped flow. Control nucleotide-induced monomer (rK340) unbinding was 73.6 s(-1) for ATP and 40.5 s(-1) for ADP. Control ADP-induced dimer (rK430) unbinding was 18.6 s(-1). Added 20 mM Pi slowed both monomer and dimer unbinding. With the roadblock in place, lead head attachment of dimers is prevented and ATP-induced trail head unbinding was then 42 s(-1). This is less than two-fold slower than the stepping rate of unimpeded rK430 dimers (50-70 s(-1)), indicating that during walking, lead head attachment induces at most only a slight (less than two-fold) acceleration of trail head detachment. As we discuss, this implies a coordination model having very fast (>2000 s(-1)) ATP-induced attachment of the lead head, followed by slower, strain-sensitive ADP release from the lead head.

Item Type: Article
DOI/Identification number: 10.1038/sj.emboj.7600042
Subjects: Q Science
Divisions: Faculties > Sciences > School of Biosciences > Protein Science Group
Depositing User: Susan Davies
Date Deposited: 19 Dec 2007 17:46 UTC
Last Modified: 23 Jan 2020 04:00 UTC
Resource URI: https://kar.kent.ac.uk/id/eprint/26 (The current URI for this page, for reference purposes)
Geeves, Michael A.: https://orcid.org/0000-0002-9364-8898
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