Chemistry Reference
In-Depth Information
Figure 3.10 Dual color FIONA, A, cartoon showing the expected
steps of a myosin V molecule labeled with bifunctional
rhodamine and Cy5 in different positions. B, FIONA recording
of rhodamine and Cy5 swapping the leading position, as
expected. A. Yildiz, P.R. Selvin and Y.E. Goldman, unpublished
observations.
evidence for the hand-over-hand mechanism and rule out hypotheses in which the
two heads do not exchange places during the step.
What causes the motion? The crystal structures of various myosin conformations
suggest that the CaM/light chain region can tilt relative to the motor domain. This
data leads to a natural hypothesis that the tilting is aworking stroke and the light chain
domain acts as a lever arm to amplify smaller structural changes in themotor domain
into the nanometer translocation of the step. Angular measurements of the myosin
V CaM subunits using polTIRF [42, 73] or DOPI [56] showed that they do rotate
approximately 70
in the plane of the actin
filament on each mechanical step
(Figure 3.11). Note the repeated increase and decrease of the axial angle
(bottom
red trace) as the myosin V lever arm adopts the leading and trailing positions during
stepping. This motion is often straight along the actin axis (in Figure 3.11, the
azimuthal angle around actin,
b
, the green trace at the bottom, is relatively constant).
In some cases, tipping around the
filament axis is also possible [56, 73, 77]. With
engineered constructs having various lengths above and below the native number of
six CaM/light chain sites, the step size of the molecule varies directly with the length
of the lever arm [67, 71, 78, 79], con
rming that the tilting of the CaM lever arm
determines the distance moved.
In native myosin V, the lever arm is approximately 24 nm long (six CaM subunits,
each 3.6 nm long plus 2.4 nm for the tilting part of themotor domain). A 70
tilt along
actinwould translate the lever arm
-
tail junction by 27.5 nm (2
a
sin (70
/2), which
is considerably less than the 36-nm step measured by FIONA, polTIRF, and the
optical trap [27, 42, 61]. Mechanical measurements on a single-headed construct
of myosin V gave
24
24 nm for the stroke size due to tilting of the lever arm [69].
