Principles of Fluorescence Spectroscopy

PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 481
Figure 14.6. Frequency-domain donor intensity decays of
DNS–(pro) 6 –TrpNH 2 (top) and DNS–(Gly) 6 –TrpNH 2 (bottom). For
the D–A pairs the dashed and solid lines are the best single and double
exponential fits, respectively.
The frequency-domain data can be analyzed to recover
the distance distribution P(r). This is accomplished by
using eq. 14.6 to predict the phase and modulation values
for assumed values of r and hw using the general procedures
described in Chapter 4 for time-domain data or Chapter
5 for frequency-domain data. These expressions are
shown below (Section 14.4) for the case of single- and
multi-exponential donor decays. Analyses of the FD data in
terms of the distance-distribution model for both peptides
are shown in Figure 14.7. These fits reveal wide and narrow
distributions for the flexible and rigid peptides, respectively
(Figure 14.8). In this analysis we used the complexity of
the donor decays (Figure 14.2, left) to recover the distribution
of D–A distances (Figure 14.2, right). The complexity
in the donor decay caused by the acceptor (Figure 14.6) was
used to determine the probability distribution of acceptors
around the donor (Figure 14.8).
14.2.2. Crossfitting Data to Exclude Alternative
Models
In addition to obtaining the average distance r and half
width hw, it is important to consider whether the data
exclude other distance distributions. This can be accomplished
by the procedure of crossfitting the data. This
Figure 14.7. Distance-distribution fits to the frequency-domain donor
decays for flexible (left) and rigid (right) peptides. The hw values in
angular brackets (< >) indicate that this value was held constant in the
analysis.
involves using one or more of the parameter values from a
second competing model to see if they are consistent with
the data. If a competing model also fits the data, then we
cannot exclude that model from our consideration without
Figure 14.8. Distance–distribution fits for DNS–(Gly) 6 –TrpNH 2
(solid) and DNS–(pro) 6 -TrpNH 2 (dashed). Revised from [7].