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Studying Kinesin’s Enzymatic Cycle Using a Single-Motor Confocal Motility Assay, Employing Förster Resonance Energy Transfer

  • Zdenek Lansky
  • Erwin J. G. PetermanEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 778)

Abstract

Kinesin is an essential eukaryotic protein that drives intracellular transport of cargo, such as vesicles and organelles. It is the smallest motor protein known that converts free energy obtained from ATP hydrolysis into mechanical work, by stepping along microtubules. The enzymatic cycle of kinesin is tightly coupled to mechanical action. How kinesin’s two identical motor domains (that both bind and hydrolyze ATP and bind to a microtubule) bring about motility has been the subject of much research. Recently, we have developed and applied a single-motor motility assay based on confocal fluorescence microscopy to measure changes in distance and orientation of the two motor domains during processive walking using Förster resonance energy transfer. The key benefit of this approach is its unprecedented time resolution of about 0.1 ms. In this chapter, we explain our approach in detailed protocols.

Key words

Kinesin FRET Single-molecule fluorescence Correlation analysis Motility assays 

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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Laboratory of Plant Cell BiologyWageningen UniversityWageningenThe Netherlands
  2. 2.Department of Physics and Astronomy and Laser CentreVU University AmsterdamAmsterdamThe Netherlands

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