Structure and dynamics of the kinesin-microtubule interaction revealed by fluorescence polarization microscopy.
Fluorescence polarization microscopy (FPM) is the analysis of the polarization of light in a fluorescent microscope in order to determine the angular orientation and rotational mobility of fluorescent molecules. Key advantages of FPM, relative to other structural analysis techniques, are that it allows the detection of conformational changes of fluorescently labeled macromolecules in real time in physiological conditions and at the single-molecule level. In this chapter we describe in detail the FPM experimental set-up and analysis methods we have used to investigate structural intermediates of the motor protein kinesin-1 associated with its walking mechanism along microtubules. We also briefly describe additional FPM methods that have been used to investigate other macromolecular complexes.
Sosa
Hernando
H
Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Asenjo
Ana B
AB
Peterman
Erwin J G
EJ
D000818:Animals; D005454:Fluorescence Polarization / Q000295:instrumentation / Q000379:methods; D006801:Humans; D016547:Kinesins / Q000737:chemistry* / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods*; D008870:Microtubules / Q000737:chemistry* / Q000378:metabolism*; D008954:Models, Biological; D011485:Protein Binding; D011487:Protein Conformation; D055503:Protein Multimerization
D016547:Kinesins
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