Biology Reference
In-Depth Information
Fig. 11.32 Single-molecule fluorescence measurements using the total internal reflection fluores-
cence (TIRF) microscopy (Axelrod 1989). (a) The basic system of illuminating single fluorescent
molecules on the glass surface (see the
black horizontal line
) using the
total internal reflection
fluorescence (TIRF) microscopy.
Typical images of single molecules measured with TIRF micros-
copy are shown (see the
white dots
in the
dark background
). (b) Visualizing the sliding movement
of a GFP (
Green
Fluorescent Protein)-tagged myosin molecule along an actin filament
immobilized on the glass surface. (c) Visualizing the turnover of ATP hydrolysis by a single
myosin molecule attached to a glass surface. Cy3-ATP and Cy3-ADP are labeled with a
fluorophore (i.e., a portion of a molecule that absorbs light at one wavelength and emits a part
of it as a longer-wavelength fluorescence; see Fig.
11.16
). Cy3-ATP binds to the myosin head and
stays bound until myosin head cleaves Cy3-ATP to produce Cy3-ADP and inorganic phosphate,
Pi. Cy3-ADP then de-binds from the myosin head due to its low binding affinity and the
fluorescent spot disappears. Thus, by using the methods employed in (b) and (c), it is possible to
measure both single myosin molecular motions and the kinetics of ATP hydrolysis accompanying
the myosin motion (see Fig.
11.33
) (Reproduced from Ihsii and Yanagida 2007))
The conformon mechanism of muscle contraction proposed in Fig.
11.31
is
supported by the data obtained from the single-molecule measurements of myosin
moving along the actin filament in the presence of ATP. Such a single-molecule
experiment was made possible because of the development of the optical (or laser)
tweezers and the total internal reflection fluorescence (TIRF) microscopy (see Figs.
11.31
and
11.32
). A 'laser tweezer', or 'optical tweezer' is a laser beam focused
down to a diffraction-limited spot of about 1
m
m in diameter. The laser beam
provides an electric field with a gradient in every direction such that there is one
point of maximum field strength. Due to the polarizing effect of the focused field,
