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

Kinesin is a molecular motor that interacts with microtubules and uses the energy of ATP hydrolysis to produce force and movement in cells. To investi gate the conformational changes associated with this mechanochemical energy conversion, we developed a fluorescence polarization microscope that allow s us to obtain information on the orientation of single as well as many flu orophores. We attached either monofunctional or bifunctional fluorescent pr obes to the kinesin motor domain. Both types of labeled kinesins show aniso tropic fluorescence signals when bound to axonemal microtubules, but the bi functional probe is less mobile resulting In higher anisotropy. From the po larization experiments with the bifunctional probe, we determined the orien tation of kinesin bound to microtubules in the presence of AMP-PNP and foun d close agreement with previous models derived from cryo-electron microscop y. We also compared the polarization anisotropy of monomeric and dimeric ki nesin constructs bound to microtubules in the presence of AMP-PNP. Our resu lts support models of mechanochemistry that require a state in which both m otor domains of a kinesin dimer bind simultaneously with similar orientatio n with respect to the microtubule.