Principles of Fluorescence Spectroscopy

PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 555
Figure 16.45. Structure of triosephosphate isomerase. Courtesy of Dr.
Hema Balaram from the Jawaharlal Nehru Center for Advanced Scientific
Research, India.
more complex cases in which there is trp-to-trp energy
transfer.
16.8.1. Single-Tryptophan Mutants of
Triosephosphate Isomerase
Triosephosphate isomerase (TPI) is a protein that catalyzes
isomerization of dihydroxyacetone phosphate and glyceraldehyde
phosphate. The enzyme from the malarial parasite
Plasmodium falciparum contains two tryptophan residues,
at positions 11 and 168. The enzyme exists as a homodimer
with each monomer containing W11 and W168 (Figure
16.45). The monomer structure consists of a central β-sheet
surrounded by α-helices, which is a common structural feature
in this class of enzymes. It is difficult to judge the
degree of exposure of each residue to water. Depending on
the viewing angle, W168 appears to be either exposed to
water (left monomer) or shielded from water (right
monomer).
The contributions of each tryptophan emission to the
total emission of TPI were determined by construction of
the single-tryptophan mutants. 138 In each mutant one of the
tryptophans was replaced with phenylalanine (F). The
W168F mutant contains only W11 and the W11F mutant
contains only W168. Emission spectra of the wild-type and
mutant proteins are shown in Figure 16.46 (top panel). The
emission from W168 is strongly blue shifted with an emission
maximum at 321 nm, and the emission maximum of
W11 is 332 nm. In this example the emission of the wildtype
protein appears to be the sum of the emission from the
two single-tryptophan mutants. This result indicates the
Figure 16.46. Emission spectra of wild-type triosephosphate isomerase
and the two single-tryptophan mutants. Revised from [138].
tryptophan residues do not interact with each other. Denaturation
of the protein in 6 M GuHCl equalized the emission
maxima of both tryptophan residues to 357 nm (lower
panel). The red shifts upon unfolding show that both
residues are shielded from water to some extent in the
native protein structure.
The emission spectra of the TPI mutants showed that
both tryptophans are sensitive to the protein structure, and
that these residues do not interact in the native protein. This
result suggests that each tryptophan residue could be used
to detect the effects of the trp-to-phe mutation on stability
of TPI. Figure 16.47 shows the effects of the denaturant
guanidine hydrochloride (GuHCl) on the emission maxima
of the wild-type TPI and the two single-tryptophan mutants.
The W168F mutant with W11 has the same stability as TPI.
In contrast, the W11F mutant with W168 denatures more
rapidly in GuHCl, which indicates lower protein stability.
Hence, substitution of W11 with a phenylalanine destabilizes
the protein. This is a surprising result because the
emission spectra (Figure 16.46) showed that W168 is buried
more deeply in the protein. One may have expected the
buried residue to contribute more to the protein stability.