Principles of Fluorescence Spectroscopy

556 PROTEIN FLUORESCENCE
Figure 16.47. Effect of guanidium hydrochloride on the wild-type
triosephosphate isomerase and the two single-tryptophan mutants.
Revised from [138].
16.8.2. Barnase:A Three-Tryptophan Protein
Barnase is an extracellular ribonuclease that is often used as
a model for protein folding. It is a relatively small protein:
110 amino acids, 12.4 kDa. The wild-type protein contains
three tryptophan residues (Figure 16.48), one of which is
located close to histidine 18 (H18). Examination of the
three single-tryptophan mutants of barnase provides an
example of energy transfer between tryptophan residues
and the quenching effects of the nearby histidine. 139
Figure 16.48. Structure of barnase showing the positions of the three
tryptophan residues and histidine 18.
Figure 16.49. pH-dependent intensity of the tryptophan emission of
wild-type barnase. Excitation, 295 nm; emission, 340 nm. Revised
and reprinted with permission from [139]. Copyright © 1991,
American Chemical Society.
The emission intensity of wild-type barnase, which
contains all three tryptophan residues, increases nearly
twofold as the pH is increased from 7 to 8.5 (Figure 16.49).
This increase in fluorescence is due to the presence of W94
and the histidine. Only mutants containing both his-18 and
trp-94 showed pH dependent intensities. Tryptophan residues
are known to be quenched by the protonated form of
histidine. 140–141 This is not surprising given the sensitivity of
indole to electron deficient molecules.
Emission spectra of barnase and its mutants at pH 5.5
and 9.4 are shown in Figure 16.50. At first glance these
spectra appear complex. First consider the contribution of
W71. Substitution of W71 with tyrosine (W71Y) has little
effect on the spectra. Hence, W71 is weakly fluorescent. It
appears that weakly fluorescent or nonfluorescent trp
residues is a common occurrence in proteins. Such effects
may contribute to the frequent observation of longer-thanexpected
lifetimes of proteins that display low quantum
yields and long apparent natural lifetimes (Figure 16.12).
The effect of H18 is immediately apparent from the
glycine mutant H18G. The intensity at pH 5.5 increases 2.6fold
over that for the wild-type protein (Figure 16.50), and
the intensity at pH 9.4 increases by about 70%. This result
indicates that both the protonated and neutral forms of histidine
quenched W94, but that quenching by the electrondeficient
protonated form present at pH 5.5 is more efficient
quencher.
Surprising results were found when W94 was replaced
with leucine in W94L. The intensity of the protein increased
even though the number of tryptophan residues
decreased. These effects were not due to a structural change