Chemistry Reference
In-Depth Information
their intensity. Binding to myosin was recognized as
fixed localization identifying
individual ATPase turnovers.
Combining TIRF microscopy with optical trap measurements of actin displace-
ments by myosin II enabled correlation of the mechanical working stroke with
binding of ATPand dissociation of ADP [96]. As expected fromsolution biochemistry,
ATP caused rapid dissociation of myosin from actin. Surprisingly, ADP often
dissociated from the myosin
-
ADP complex before it rebound to actin, leading to
the suggestion that the energy liberated by ATP hydrolysis can be stored in the protein
even after the nucleotide is released. This apparent protein hysteresis has been
detected in other enzymes [97, 98].
The angle of the myosin II light chain region has been measured during the
working stroke in bulk assays within muscle
fibers using
fluorescence polariza-
tion [41, 99] and low angle X-ray diffraction [100]. This structural change takes place on
the 1
-
5-ms timescale. Whether the average structural signals from these bulk assays
faithfully represent the behavior of the individual molecules is an important applica-
tion for single molecule imaging. Angular motions have been made by single
molecule
fluorescence polarization [15, 101]. The limited lifetime of the actomyosin
complex, however, and the rapid kinetics of the myosin II working stroke have so far
challenged singlemolecule detection of the angular change during its working stroke.
3.3.3
Myosin VI
This molecular motor, involved in cargo transport, endocytosis and anchoring in
neural development and in sensory cells, exhibits several interesting and puzzling
features [102]. Unlike the other characterized myosins which translate toward the
barbed end of the actin
filament, myosin VI moves toward the pointed end [103, 104]
due to a peptide insert in the motor domain that orients the lever arm opposite to the
other myosins [105]. The directionality can be determined using actin
filaments
whose polarity is marked by a bright region of
fluorescent actin or a speci
c
fluorescent actin-binding protein [103, 105]. Polarity marked
filaments show the
direction of the active motion in both the single-molecule TIRF processivity assay
(myosin walks on actin
filaments attached to the substrate) and the gliding
filament.
assay (myosin is attached to the surface and moves the actin).
Double-headed molecules of myosin VI are processive with highly variable step
sizes ranging from 20 to 40 nm and display many more backward steps than myosin
V [106]. The distancemyosin VI can traverse per mechanical step is surprisingly large
because it contains only two CaMs per putative lever arm, compared to six per lever
arm in myosin V. Whether the motor is a dimer or monomer within cells is
controversial [107]. There is evidence that cargos or actin can facilitate dimeriza-
tion [108, 109] and that even a single head canmove processively when it is attached to
a bead cargo [109]. As with myosin V, processive motility by single-headed constructs
has not been reported using single-molecule
fluorophore imaging, possibly
indicating that an attached bead enhances processivity by restricting diffusion away
from the
filament.
