is a dynamic structure that can adopt two different conformations that
are in equilibrium with each other. In one conformation the distance between
the energy donor and acceptor is large and energy transfer cannot occur,
but in the other conformation the distance is small and energy transfer
does occur.
In a bulk measurement of the Holliday junction the presence of the
different conformations cannot be distinguished since all states are sampled
simultaneously and averaged. Single-molecule FRET measurements allow
the measurement of the distance between the donor and acceptor
from a single molecule. As the conformation switches back and forth, the
fluorescence will be measured from either the donor, indicating that
the donor–acceptor distance is large, or the acceptor, indicating that the
donor–acceptor distance is small (Figure 14.23). In these experiments, a
variable time interval is evident between
conformational switching because the process is stochastic. That is, the switching
occurs with a certain probability only, and
so each time the period is different, and
only the average time must agree with bulk
measurements of the switching rate.
FRET can also be used to study interactions between different parts of DNA
due to protein binding (Katilitene et al.
2003). For example, some DNA restriction
endonucleases, such as NgoMIV, function
by binding simultaneously at two specific
recognition sites, making a DNA loop,
314
PART 2
QUANTUM MECHANICS AND SPECTROSCOPY
(a)
(c)
(b)
Cy3
Cy3
Cy5
Cy5
Cy5
Cy3
Figure 14.22 The different conformations of the Holliday junction showing the different relative
positions of the energy donor and acceptor. (a) A conformation with a limited FRET signal,
(b) a proposed intermediate, and (c) an alternate conformation with a strong FRET signal.
Modified from Churchill (2003).
0.5
0.0
0
1
2
3
4
5
6
7
FRET
Time (s)
Figure 14.23 The measurements of the Holliday
junction give a signal from either the energy
acceptor, with a high FRET value, or from the
energy donor, with a low FRET value. Modified
from Churchill (2003).
9781405124362_4_014.qxd 4/29/08 13:09 Page 314
are in equilibrium with each other. In one conformation the distance between
the energy donor and acceptor is large and energy transfer cannot occur,
but in the other conformation the distance is small and energy transfer
does occur.
In a bulk measurement of the Holliday junction the presence of the
different conformations cannot be distinguished since all states are sampled
simultaneously and averaged. Single-molecule FRET measurements allow
the measurement of the distance between the donor and acceptor
from a single molecule. As the conformation switches back and forth, the
fluorescence will be measured from either the donor, indicating that
the donor–acceptor distance is large, or the acceptor, indicating that the
donor–acceptor distance is small (Figure 14.23). In these experiments, a
variable time interval is evident between
conformational switching because the process is stochastic. That is, the switching
occurs with a certain probability only, and
so each time the period is different, and
only the average time must agree with bulk
measurements of the switching rate.
FRET can also be used to study interactions between different parts of DNA
due to protein binding (Katilitene et al.
2003). For example, some DNA restriction
endonucleases, such as NgoMIV, function
by binding simultaneously at two specific
recognition sites, making a DNA loop,
314
PART 2
QUANTUM MECHANICS AND SPECTROSCOPY
(a)
(c)
(b)
Cy3
Cy3
Cy5
Cy5
Cy5
Cy3
Figure 14.22 The different conformations of the Holliday junction showing the different relative
positions of the energy donor and acceptor. (a) A conformation with a limited FRET signal,
(b) a proposed intermediate, and (c) an alternate conformation with a strong FRET signal.
Modified from Churchill (2003).
0.5
0.0
0
1
2
3
4
5
6
7
FRET
Time (s)
Figure 14.23 The measurements of the Holliday
junction give a signal from either the energy
acceptor, with a high FRET value, or from the
energy donor, with a low FRET value. Modified
from Churchill (2003).
9781405124362_4_014.qxd 4/29/08 13:09 Page 314