Article
- The EMBO Journal (2004) 23, 23 - 32
- doi:10.1038/sj.emboj.7600042
Published online: 18 December 2003
Subject Category:
What kinesin does at roadblocks: the coordination mechanism for molecular walking
Isabelle M-TC Crevel1,a, Miklós Nyitrai2,3,a, María C Alonso1, Stefan Weiss2, Michael A Geeves2 and Robert A Cross1
- Molecular Motors Group, Marie Curie Research Institute, The Chart, Oxted, Surrey, UK
- Department of Biosciences, University of Kent, Canterbury, Kent, UK
- Research Group for Fluorescence Spectroscopy, Office for Academy Research Groups Attached to Universities and Other Institutions, Department of Biophysics, Faculty of Medicine, University of Pécs, Pécs, Hungary
Correspondence to:
Robert A Cross, Molecular Motors Group, Marie Curie Research Institute, The Chart, Oxted RH8 0TL, Surrey, UK. Tel.: +44 1883 722306; Fax: +44 1883 714 375; E-mail: r.cross@mcri.ac.uk
aThese authors contributed equally to this work
Received 1 August 2003; Accepted 17 November 2003
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.
Keywords:
- kinesin,
- kinesin mechanism,
- processivity,
- tubulin
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