Chemistry Reference
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FIONA experiments showed that the individual heads of the myosin VI dimer
translate by 70 nm on each step [34, 110], implying a hand-over-hand mechanism, as
with myosin V. As the lever arms are too short for the molecule to span over the
corresponding 35-nm distance between target actin sites, asymmetric models of the
motion were considered in which the motor or dimerization domains would partially
unfold to enhance the molecules reach. Such a difference between the two heads
might result in two sets of distances between the heads corresponding to the
unfolded one leading or trailing. Measurement of the distance between two GFP-
labeledmyosinVI heads by SHRImP showed this not to be the case (Figure 3.5) [111].
A strongly dimerizing construct of myosin VI was expressed with a GFP tag on each
head. When this species was bound to actin and imaged using TIRF microscopy,
sequential bleaching of the fluorophores on each of the heads, and subtraction of the
one- and two- uorophore PSF distributions (Figure 3.5) gave the inter-probe
distance. For myosin VI, the distance between the two heads bound to actin was
found to be 29 nm with an upper limit of
14 nm for any supposed asymmetry
(e.g. two distances 22 nm and 36 nm). These data are consistent with a symmetrical
hand-over-hand stepping mechanism.
Optical trap experiments [112] and X-ray crystallography [113] have suggested
that the lever arm rotates nearly 180 during the working stroke of myosin VI. With
a 10-nm long lever arm, the maximum rotational working stroke is 20 nm,
necessitating a diffusional search as in myosin V [110, 112]. A polTIRF study of
myosin VI dimers detected large rotational motions of the CaM during processive
stepping [52], with a highly variable angle when the head binds actin in the leading
position. Taken together with the high variance of mechanical step distance [106]
and leading position [110], these experiments suggest that the thermal search
reaches actin monomers having a large range of axial distances and azimuthal
angles around the actin axis. Due to the structure of the actin filament, binding of
the leading head to actins 6, 7, 9, 11 and 13 monomer units away from the attached
head require sideways tilting of various amounts up to nearly a
90 azimuth [52].
This wide choice of target actin monomers may enable myosin VI to navigate
around obstacles and to switch actin
filaments in order to carry out cargo transport
functions in the crowded cellular environment. Investigations into the kinetic
coupling between the two heads suggest that intramolecular strainmodulates both
ADP release and ATP binding kinetics [114, 115], another novel mechanism in this
unusual motor.
The description here of myosin experiments has emphasized points that are
reasonably agreed upon in order to illustrate the advances made by imaging single
fluorescent probes. But there are many other issues that are not settled and will be
approached by single molecule biophysics. How is binding of the myosin head to
actin coupled to the working stroke and dissociation of Pi i and ADP? How is the path
along actin determined? How often do backward steps occur and what is their
signi cance and biochemical pathway? Is there a torque associated with the working
stroke? Does the tilting of the lever arm power the motion or just sense it? Do single-
headed myosins work processively? What novel mechanisms will be revealed by
study of the almost 20 myosin family members that have hardly been studied by
 
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