BioPHYSICAL chemistry

In terms of experimental information, the rate can be expressed using a
number of parameters. Most critical is the extent of the spectral overlap.
For the energy transfer to occur, the energy difference from D to D must
match that from A to A. In spectral terms, the donor emission band must
overlap with the acceptor absorption band (Figure 19.7). The second
critical factor is the separation between the donor and acceptor, with the
rate decreasing as r−^6. For many experiments, the measurement is per-
formed under steady-state conditions and so the data provide not the
rate of energy transfer by rather the efficiency of transfer, which can be
expressed simply in terms of the separation:

(19.3)

The distance dependence for the efficiency is seen to result in a sharp
dependence centered on the parameter r 0 that represents the distance
at which the efficiency of energy transfer is 50%. This constant can be
written in terms of the various nondistance factors such as the spectral

Ef ciency of transfer
DA

1 =

+

r

rr

0

6

0

66

CHAPTER 19 MOLECULAR IMAGING 411

R  1.5R 0

Donor

excitation

(a)

Donor

(GFP)

Donor

emission

Acceptor

(Cy3)

Donor

excitation

Protein B

(dye-labeled

substrate)

Protein B

(enzyme–GFP)

FRET

‘dark process’

Acceptor

‘sensitized’

emission

R  1.5R 0

(c)

Wavelength (λ)

Overlap

region

Donor

abs

Donor

em

Acceptor

abs

Acceptor

em

1.0

0.5E

0.0

0.0

(b)

1.0 2.0 3.0

r (units of R 0 )

Figure 19.7A FRET experiment can be used to measure the
relative number of proteins forming complexes. (a) In both
cases, the GFP is excited but subsequent emission arises from
either the donor, when the proteins are far apart, or from the
acceptor, when the proteins form a complex. The efficiency
of the energy transfer has a sharp dependence upon (b) the
distance and (c) requires a substantial spectral overlap. abs,
absorption; em, emission. Modified from Bastiaens and
Pepperkok (2000).