Principles of Fluorescence Spectroscopy

452 ENERGY TRANSFER
safe for many proteins in the native state, particularly for
single-domain proteins. For unfolded peptides or multidomain
proteins a variety of conformations can exist, which
results in a range of D–A distances. In this case calculation
of a single distance using eq. 13.12 would result in an
apparent distance, which would be weighted towards the
shorter distances. Such systems are best analyzed in terms
of a distance distribution using the time-resolved data
(Chapter 14).
13.3.2. Effects of Incomplete Labeling
The largest source of error in calculating distance from the
RET data is probably incomplete labeling with the acceptor.
If melittin were incompletely labeled with acceptor, the
measured value of F DA would be larger than the true value,
and the calculated distance too large. We are less concerned
with underlabeling by the donor because the protein molecules
that do not contain donors do not contribute to the
donor intensity, assuming the extent of donor labeling is the
same for the donor-alone and donor–acceptor pair.
If the fractional labeling with donor (f a ) is known then
the relative intensities can be used to calculate the transfer
efficiency. In this case eq. 13.14 becomes 13
E 1 F DA F D(1 f A)
F Df A
( 1 FDA ) FD 1
fA (13.17)
For a high degree of RET donor quenching, (F DA /F D