Published in

Elsevier, Biophysical Journal, 5(81), p. 2851-2863, 2001

DOI: 10.1016/s0006-3495(01)75926-7



Export citation

Search in Google Scholar

Polarized fluorescence microscopy of individual and many kinesin motors bound to axonemal microtubules.

This paper is made freely available by the publisher.
This paper is made freely available by the publisher.

Full text: Download

Green circle
Preprint: archiving allowed
Orange circle
Postprint: archiving restricted
Red circle
Published version: archiving forbidden
Data provided by SHERPA/RoMEO


Kinesin is a molecular motor that interacts with microtubules and uses the energy of ATP hydrolysis to produce force and movement in cells. To investigate the conformational changes associated with this mechanochemical energy conversion, we developed a fluorescence polarization microscope that allows us to obtain information on the orientation of single as well as many fluorophores. We attached either monofunctional or bifunctional fluorescent probes to the kinesin motor domain. Both types of labeled kinesins show anisotropic fluorescence signals when bound to axonemal microtubules, but the bifunctional probe is less mobile resulting in higher anisotropy. From the polarization experiments with the bifunctional probe, we determined the orientation of kinesin bound to microtubules in the presence of AMP-PNP and found close agreement with previous models derived from cryo-electron microscopy. We also compared the polarization anisotropy of monomeric and dimeric kinesin constructs bound to microtubules in the presence of AMP-PNP. Our results support models of mechanochemistry that require a state in which both motor domains of a kinesin dimer bind simultaneously with similar orientation with respect to the microtubule.