Chemistry Reference
In-Depth Information
Figure 3.11 PolTIRF recordings from a
bifunctional rhodamine labeled calmodulin
exchanged into myosin V. The motor is walking
processively along actin at 5
polarizations as defined in the Figure 3.6. The
sum of the polarized intensities is plotted as
calc; fitted total intensity, less background, is
blue. The single discrete decrease of all
intensities to background levels at 3.7 s is due
to photo-bleaching of the fluorophore.
From Ref. [42].
M MgATP.
Polarized fluorescence intensities are given as
photocounts per 10-ms gate. Polarized intensity
subscripts indicate excitation/detection
m
Those authors hypothesized that the remainder of the 36-nm step of the whole,
double-headed molecule is accomplished by thermal
fluctuations of a singly-bound
intermediate randomly carrying the free head to the actinmonomer 36 nm in front of
the stationary head.
Using the dark
eld adaptation of FIONA explained above (DIONA) to make a
major improvement to the time resolution, Dunn and Spudich [36] were able to detect
the singly-bound state expected from the diffusional search model. A 40-nm gold
nano-particle was attached to a CaM subunit and viewed by scattered light under
highly oblique, propagating illumination (Figure 3.12). A transient intermediate
position with
fluctuations was evident (horizontal arrows in panel B), con
rming the
random search hypothesis for completion of the step.
This remarkable behavior implies that themolecule is harnessing random thermal
fluctuations to extend its reach. No thermodynamic laws are violated here in making
fluctuations useful because the energy liberated in the ATPase cycle enables the
overall process to proceed in the forward direction [80]. When the wobbling head in
