BioPHYSICAL chemistry

is a dynamic structure that can adopt two different conformations that

are in equilibrium with each other. In one conformation the distancebetween

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

variabletime interval is evident between

conformational switching because the pro-

cess 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 inter-

actions 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) Cy3 (b) (c)

Cy3

Cy5

Cy5

Cy5

Cy3

Figure 14.22The 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

01234567

FRET

Time (s)

Figure 14.23The 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).