Crevel, I.M.-T.C. and Nyitrai, M. and Alonso, M.C. and Weiss, S. and Geeves, M.A. and Cross, R.A. (2004) What kinesin does at roadblocks: the coordination mechanism for molecular walking. EMBO Journal, 23 (1). pp. 23-32.
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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.
|Divisions:||Faculties > Science Technology and Medical Studies > School of Biosciences > Protein Science Group|
|Depositing User:||Sue Davies|
|Date Deposited:||19 Dec 2007 17:46|
|Last Modified:||05 Sep 2011 23:19|
|Resource URI:||http://kar.kent.ac.uk/id/eprint/26 (The current URI for this page, for reference purposes)|
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