Chemistry Reference
In-Depth Information
2.3.1
Observation and Manipulation of a Single Myosin Motor
Myosin heads were labeled at their tail ends with a
fluorescent dye (Cy3) in an almost
one to one (0.95) molar ratio. The number of myosin heads captured onto the probe
tip was determined from the
fluorescence intensity and photobleaching behavior.
The
-
labeled myosin could be clearly observed as
fluorescent spots using evanescent
eld
illumination. The Cy3
uorescence was observed by an objective type TIRFM [4] (Figure 2.4a). The Cy3
labeledmyosinwas captured at its biotinylated tail on the tip of
a probe through a biotin
-
streptavidin bond. Figure 2.4b shows typical
uorescence
time trajectories of Cy3
-
labeled myosin captured on the tip of the probe in which the
fluorescence intensities decreased in a single step (upper) and double step (lower).
The intensity of the
uorescent spots of single Cy3-labeledmyosin captured on the tip
of the probe was the same as that of those bound to the glass. These results indicate
that it is possible to count the number of S1 molecules on the tip of the probe. For
example, the number of Cy3
-
BDTC
-
S1 molecules on the tip of the probe in
Figure 2.4b (upper and lower panels) was judged to be one and two, respectively.
Only the data for single molecules were used for the following analysis.
-
2.3.2
Displacements
To minimize damage to the myosin caused by interaction with the surface of the
probe, the myosin was speci
cally attached to the tip of a probe at its tail end via a
biotin
streptavidin bond. After the number of myosins on the probe tip was
-
con
fluorescence, the captured myosin head was brought into
contact with an actin bundle
fixed to the glass surface in the presence of ATP
(Figure 2.4a). Displacements produced by singlemyosins weremeasured with a wide
range of needle stiffness from 0.01 to 0.6 pN/nm.
Figure 2.5a and b (upper traces) show typical time courses of displacements at low
and high needle stiffness. The myosin
-
actin interactions could be clearly identi
ed
by an increase in stiffness calculated from the reciprocal of the variance of the
fluctuations of the probe. Thermal
fluctuations occurred when myosin dissociated
from the actin bundle. Their amplitude was dependent on the stiffness of the probe.
During myosin
-
actin attachments, the
fluctuations decreased to an r.m.s. amplitude
of 1.4
-
2.9 nmwhich corresponded to a stiffness of 0.5
-
2 pN/nm. The highest value of
stiffness during attachments (
rmed to be one by
2 pN/nm) was as large as that of an actomyosin
crossbridge in muscle [26].
The mean displacement of myosin was determined by averaging observed events
(n
274). The mean displacement was 17 nm at low needle stiffness (0.01
-
0.1 pN/
nm) and 9.2 nm at high needle stiffness (0.1
-
0.6 pN/nm). The duration of displace-
ments in the presence of 1mM ATP at 20
C was distributed exponentially. The
second-order rate constant for the dissociation of actin
-
myosin by ATP was deduced
from the mean duration and found to be 4
-
5
ΒΌ
10
6
M
1
s
1
at low needle stiffness.
This value was consistent with the values obtained for the suspension of actin and
