Principles of Fluorescence Spectroscopy

560 PROTEIN FLUORESCENCE
three residues. For instance, the structure surrounding
W248 persists to higher guanidium concentrations than the
structured around residues W279 and W285. From such
data it is possible to reconstruct which regions of the protein
are more stable, and which are first disrupted during
protein unfolding.
16.9.3. Folding Pathway of CRABPI
The folding pathway and kinetics can be studied using
mutant proteins. 160–161 This was accomplished using cellular
retinoic acid binding protein I (CRABPI). This protein
has three tryptophan residues at positions 7, 87, and 109
(Figure 16.57). In the native state the emission spectra are
strongly dependent on the excitation wavelength (not
shown). This occurs because each of the tryptophan
Figure 16.57. Structure (top) and refolding kinetics (bottom) of the
three single-tryptophan mutants of CRABPI. Courtesy of Dr. Lila M.
Gierasch from the University of Massachusetts.
residues are present in different environments, and the
absorption and emission spectra of the residues depend on
the local environment. When the protein is denatured, the
emission spectra becomes independent of the excitation
wavelength because the three tryptophan residues are all in
a similar environment.
CRABPI mutants were used to study refolding of the
denatured protein. This was accomplished by initially denaturing
the protein in urea. The urea was then rapidly diluted
in a stopped-flow instrument, followed by measurement
of the tryptophan emission (Figure 16.57). These traces
show that the region surrounding W7 folds most rapidly,
followed by folding of the regions around W87 and W109,
which occurs several-fold more slowly.
16.10. PROTEIN STRUCTURE AND
TRYPTOPHAN EMISSION
Numerous protein structures are known and it is now
becoming possible to correlate the environment around the
tryptophan residues with their spectral properties. One
example is human antithrombin (Figure 16.58). Human
antithrombin (AT) responds to heparin with a 200- to 300fold
increase in the rate of inhibition of clotting factor Xa.
Wild-type AT contains four tryptophan residues. The contri-
Figure 16.58. Structure of human antithrombin showing the four tryptophan
residues and serine residue present in the wild-type protein.
Courtesy of Dr. Peter G. W. Gettins from the University of Illinois.