Publication Date:
2009-08-21
Description:
The dimeric motor protein kinesin-1 converts chemical energy from ATP hydrolysis into mechanical work used to transport cargo along microtubules. Cargo attached to the kinesin stalk moves processively in 8-nm increments as its twin motor domains (heads) carry out an asymmetric, 'hand-over-hand' walk. The extent of individual head interactions with the microtubule during stepping, however, remains controversial. A major experimental limitation has been the lack of a means to monitor the attachment of an individual head to the microtubule during movement, necessitating indirect approaches. Here we report the development of a single-molecule assay that can directly report head binding in a walking kinesin molecule, and show that only a single head is bound to the microtubule between steps at low ATP concentrations. A bead was linked to one of the two kinesin heads by means of a short DNA tether and used to apply rapidly alternating hindering and assisting loads with an optical trap. The time-dependent difference between forwards and backwards displacements of the bead alternated between two discrete values during stepping, corresponding to those intervals when the linked head adopted a bound or an unbound state. The linked head could only rebind the microtubule once ATP had become bound to its partner head.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2859689/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉 〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2859689/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Guydosh, Nicholas R -- Block, Steven M -- GM51453/GM/NIGMS NIH HHS/ -- R01 GM051453/GM/NIGMS NIH HHS/ -- R01 GM051453-15/GM/NIGMS NIH HHS/ -- England -- Nature. 2009 Sep 3;461(7260):125-8. doi: 10.1038/nature08259. Epub 2009 Aug 19.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Biophysics Program, Stanford University, Stanford, California 94305, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/19693012" target="_blank"〉PubMed〈/a〉
Keywords:
Adenosine Triphosphate/metabolism/pharmacology
;
Animals
;
DNA/chemistry/metabolism
;
Drosophila melanogaster
;
Kinesin/*chemistry/*metabolism
;
Microspheres
;
Microtubules/*metabolism
;
Movement/drug effects
;
Optical Tweezers
;
Protein Binding/drug effects
;
Time Factors
Print ISSN:
0028-0836
Electronic ISSN:
1476-4687
Topics:
Biology
,
Chemistry and Pharmacology
,
Medicine
,
Natural Sciences in General
,
Physics
