Chemistry Reference
In-Depth Information
dependent binding of GroES to GroEL immobilized onto a mica surface in an end-
up orientation [19]. Additionally, they observed the formation and dissociation
of a streptavidin-biotinylated DNA complex [20] and the one-dimensional
diffusion of a restriction enzyme along a DNA strand followed by DNA
cleavage [21].
The dynamic processes mentioned above were already known or expected from a
series of biochemical and biophysical studies, meaning these
filmed images did not
offer extremely new insights into the respective molecular mechanisms. Neverthe-
less, because this technique is new and unproven, known or expected biological
processes need to be con
rmed by high-speed AFM imaging before applying the
technique to broader studies. In addition, techniques to prepare samples and
substrates for their attachment need to be developed. These are often different from
their static imaging counterparts. Upon proving its utility for known molecular
processes, high-speed AFM will become a reliable tool for unexplored biological
processes.
Using high-speed AFM, we have been seeking to image single myosin V
molecules walking along actin
filaments. Single myosin V molecules move proces-
sively along actin tracks [22]. The hand-over-hand walking of myosin V is already
established [23
-
26] but its detailed behavior is still unknown. For AFM studies, the
myosin V tail was removed by digestion [27] because it tended to attach to the mica
surface. However, in a low ionic solution, the truncated myosin V (HMM) still
tended to attach to the mica surface. Therefore, we elevated the ionic strength,
although this lowered the myosin V af
nity for actin. Due to the weak af
nity, the
oscillating cantilever tip with the usual free amplitude (
5 nm) disturbed the
actin
-
myosin V interaction. Therefore, we reduced the free amplitude to
1 nm,
sacri
cing the feedback bandwidth. Under these conditions, we successfully
captured the walking movement of myosin V on video at 0.1 s/frame and
observed that during the process the leading and trailing heads altered their
positions with a walking stride of
72 nm (N. Kodera et al., unpublished data).
However, some level of myosin V af
nity for the mica surface still remained in
the high-ionic solution. Its af
nity acted as a mechanical load against myosin V
walking, and therefore, reduced the probability of observing myosin V moving
processively.
We have also been seeking to image GroEL
-
GroES interaction dynamics in an
ATP-containing solution where biotinylated GroEL [28] is immobilized on strepta-
vidin 2D-crystal sheets in a side-on orientation. Due to this orientation, both the
GroEL rings were accessible to GroES
floating in the solution. Because
floating
GroES did not interfere with imaging, a high concentration of GroES could be used,
in contrast to the situation with single-molecule
fluorescence microscopy.
The negative cooperativity between the two GroEL rings (e.g. [29]) was con
rmed.
GroEL alternated its rings between the GroES-associated and -dissociated states.
However, interestingly, releasing one GroES-associated complex and forming
another did not necessarily occur simultaneously. Two controversial intermediates
(e.g. [30]), bare GroEL and GroEL
-
(GroES)
2
, were detected just prior to the switching
(D. Yamamoto et al., unpublished data).
