Principles of Fluorescence Spectroscopy

564 PROTEIN FLUORESCENCE
Figure 16.66. Structures of tryptophan analogues. The numbers under
the structures are the quantum yields in neutral buffer [168].
using tryptophan or amino-acid analogues that absorb at
longer wavelengths than tryptophan. These analogues must
be inserted into the protein sequence. This can be accomplished
in three ways. The entire protein can be synthesized
Figure 16.67. Absorption (top) and emission spectra (bottom) of tryptophan
analogues. Revised from [168].
de novo. However, this approach is limited to small peptides
and proteins that will fold spontaneously after synthesis.
The second approach is by incorporation into protein
grown in bacteria or a cell-free system. This is typically
done using tryptophan auxotrophs, which cannot synthesize
their own tryptophan. The amino-acid analogue is chemically
attached to the tRNA, which is then added to the sample
during protein synthesis. The inserted amino acids are
typically selected to have a structure similar to tryptophan.
The third and most elegant approach is modification of the
genetic code and protein synthesis machinery to include a
new amino acid.
16.11.1. Tryptophan Analogues
A group of tryptophan analogues have been synthesized
and incorporated into proteins using tryptophan auxotrophs
168–181 (Figure 16.66). These analogues were designed
to retain a size close to tryptophan itself. Except for 4-fluorotryptophan
(4FW) these analogues absorb at longer wavelengths
than tryptophan (Figure 16.67).
The spectral properties of 5HW and 7AW are different
from tryptophan. 178 In water, 5HW displays a higher quantum
yield than tryptophan (0.275 for 5HW versus 0.13 for
W). 5HW is less sensitive to solvent polarity than tryptophan,
and displays an emission maximum near 339 nm
(Figure 16.67). The quantum yield of 7AW is highly
dependent on solvent polarity and decreases upon contact
with water. 182 In water its quantum yield is low, near 0.017,
with an emission maximum near 403 nm. This property of
7AW is somewhat problematic. The use of 7AW was originally
proposed as an alternative to tryptophan because 7AW
was thought to display a simple single exponential decay.
183–184 Unfortunately, 7AW and azaindole display complex
decay kinetics due to the presence of several solvated
states. 185–187 A non-exponential decay has also been
observed for an octapeptide that contains a 7AW residue. 188
7AW is a useful tryptophan analogue, but it can display
complex decay kinetics.
These tryptophan analogues have been incorporated
into a number of proteins. One example is substitution of
5HW and 7AW for the single-tryptophan residue in staphylococcal
nuclease (Figure 16.68). In this case the emission
maxima of tryptophan and 5HW are similar. The advantages
of 5HW and 7AW can be seen by their use in studies
of more complex biochemical mixtures. 5HW was used to
replace the tyrosine residue in insulin. This allowed the fluorescence
of 5HW-insulin to be used to study its binding to