Chemistry Reference
In-Depth Information
wavelength probe (Cy3) can be transferred to excite the longer wavelength probe
(Cy5) without radiation of an intermediate photon. The FRET technique is well
developed at the single molecule level, especially for nucleic acid processing
enzymes. It is explained in more detail in Chapter 11 of this topic. For example,
when Cy3, termed the donor, is illuminated with 514-nm laser light, instead of
fluorescing at its 570 nm emission wavelength, its excitation energy can sometimes
be transferred to nearby Cy5 probes (the acceptors). The energy transfer ismonitored
by Cy5
fluorescence at 670 nm. After correction for various spectral properties and
cross-over between the excitation and detector channels, a quantity termed FRET
ef
I
A
), where I
D
and I
A
are the donor and acceptor
fluorescence intensities. Among other factors, E depends on the distance, r, between
the two
fluorophores in the useful range of 2.5
-
7.5 nm according to E
ciency is calculated, E
¼
I
A
/(I
D
þ
r
0
=ð
r
6
r
0
Þ
¼
þ
where r
0
is the distance at which E
5.3 nm for the Cy3
-
Cy5 pair.
An example single molecule FRET experiment with kinesin is given in Figure 3.13
and explained later in the chapter.
¼
0.5, which is r
0
¼
3.2.6
Orientation of Single Molecules
Tilting and rotations of molecular domains are crucial motions in themechanisms of
many protein and RNA enzymes [39, 40]. Spatial orientation of protein domains are
especially important inmolecular motor research because the key structural changes
leading to movement are commonly attributed to tilting of protein domains, as
described below for myosin and kinesin. Quantifying angle changes in ensemble
systems, though, can be very dif
cult. For example, during contraction of muscle
fibers, most of the myosin heads are not attached to actin and have nearly random
angular distributions [41]. Concerted rotational motions among the small proportion
that are attached to actin are dif
cult to detect against the disordered background.
Single-molecule
cient
sensitivity to detect absolute orientation and tilting on the millisecond time scale
relevant to the function of the molecular motor. A bright extrinsic
fluorophore is
inserted into the structure by site-speci
c labeling and changes in the orientation of
the
fluorophore are considered to signal the rotational motions of the macromole-
cular domain. When the orientation of the probe is known relative to the attached
domain, the absolute orientation in space can be estimated [42, 43]. The relative
orientation between the probe and the protein can be preprogrammed into the
structure by the placement of the labeling sites [42, 43] or it can be determined by
crystallography [44], NMR spectroscopy [45] or molecular dynamics calculations [46].
Single molecule measurements of orientation and tilting motions have been
reviewed in [39, 40, 47, 48].
Three related physical properties of a
uorescent dipole enable determination of its
spatial orientation: (1) the relative absorption of light polarized in various directions,
(2) the angular directions of its emitted photons, and (3) their polarization. The
likelihood that a chromophore will absorb a photon is proportional to cos
2
fluorescence microscopy, on the other hand, provides suf
q
a
, where
q
a
is the angle between the photons polarization (direction of the oscillating electric
