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singlemolecule techniques? Domultiplemotors cooperate duringmotion of a cargo?
The methods described here are poised to address these questions.
3.3.4
Conventional Kinesin
The founding member of the kinesin family (Figure 3.8, kinesin-1) transports a
variety of cargos in cells, including membranous organelles, mRNA, and signaling
molecules. It is essential for mitosis and is the predominant molecular motor that
conducts antegrade transport of vesicular cargos in nerve axons. Mutations have been
linked to neurological diseases [116]. Kinesin-1 a highly processive, dimeric motor,
whose 8-nm steps toward the plus (faster polymerizing) end of microtubules match
the spacing of the tubulin dimers (Figure 3.8). An early TIRF microscopic assay
showed that Cy3-labeled dimeric kinesins moved an average of 600 nm along
microtubules attached to themicroscope slide [117], con
rming the high processivity
of kinesins mechanism found earlier in gliding
filament and optical trap stud-
ies [118, 119]. A 600-nm processive run requires completion of 75 8-nm steps before
the molecule dissociates. Monomeric constructs are not processive, indicating that
the two heads in the native molecule cooperate to maintain contact with the
microtubule during stepping. FIONA experiments, using kinesin labeled on the
motor domain with Cy3, gave alternating 17-, 0-, 17-nm steps indicating hand-over-
hand motility [120 4] in which the trailing head detaches, passes by the attached head
moving forward by a distance twice the step size, and becomes the leading head.
Themotor domains of a dimeric kinesinmolecule are too close to the junctionwith
the coiled-coil tail to accommodate a stiff lever arm that could produce an 8-nmstroke
(Figure 3.8). In crystal structures of the dimeric molecule, the two heads are only
5 nm apart raising the issue of how both motor domains can bind to tubulin dimers
separated along the microtubule by 8 nm [121]. There is a 15-amino acid sequence at
the C-terminus of the motor domain, just before the coiled-coil tail, that was found to
dock and undock from the motor domain in various steps of the ATPase cycle [122].
Ensemble distance measurements between
uorescent probes in this neck linker
and the motor domain, as well as cryo-EM and EPR studies supported its role in
generating the working stroke and enabling two-headed attachment of kinesin to
successive tubulin dimers.
A single molecule measurement, using FRET to measure intramolecular
distance [123] (Figure 3.13), enabled the structural changes in the neck linker to
be correlated with active processive motility. Engineered cysteine residues in the
motor domain (amino acid 215) and in the tail (amino acid 342) were labeledwith Cy3
and Cy5. The distance between them was estimated using smFRET as described
above. The FRETef
ciency oscillated between E
0.4 and 0.9, values consistent with
the 2-nm and 6-nm distance between the probes expected on the basis of neck linker
docking and undocking (Figure 3.13A). The docked and undocked con
gurations
could be assigned to trailing and leading positions and the rate of switching between
the high and low E states was consistent with one structural change per 8-nm step of
motility. These results link changes of the motor domain
-
neck linker interaction to
ΒΌ
