Principles of Fluorescence Spectroscopy

540 PROTEIN FLUORESCENCE
Figure 16.16. Fluorescence spectra of apo Pae azurin, apo Ade azurin,
apo Afe azurin. The fluorescence spectra of the holoproteins only differ
from that of the apoproteins in that they have a reduced intensity.
Data reprinted with permission from [79]. Copyright © 1987, American
Chemical Society.
50,000 daltons. Such a protein contains approximately 450
amino-acid residues (about 110 daltons per amino-acid
residue), about 10 of which will be one of the aromatic
amino acids. Using a typical density of proteins near 1.4
g/ml, one can calculate a protein radius near 24 Å. The concentration
of aromatic amino acids in this protein is near
0.28 M = 280 mM.
Typical Förster distances (R 0 ) and characteristic concentration
values (A 0 ) for energy transfer between the aromatic
amino acids are listed in Table 16.1. These values
illustrate the range of distances and concentrations typical
of RET between the fluorescent amino acids. For any given
donor–acceptor pair the actual distances will depend on the
quantum yield and emission spectrum of the donor, which
can vary in different protein environments. Absorption
spectra are typically less sensitive to the environment, so
the emission spectrum and quantum yield of the donor are
the dominant origin of the range of R 0 values. From the values
in Table 16.1 it is evident that RET can be expected
between the aromatic amino acids in proteins.
The Förster distance for tryptophan-to-tryptophan
homotransfer is particularly variable. This is because the
extent of spectral overlap is strongly dependent on solvent
polarity. In polar solvents the emission spectrum of tryptophan
is shifted away from the absorption spectrum and the
Förster distances are smaller. For instance, for the fully
exposed tryptophan residues in melittin the Förster distance
was estimated to be just 4 Å. 80–81 At low temperature in vis-
Table 16.1. Förster Distances and Critical Concentrations
for Resonance Energy Transfer in Proteins
Donor Acceptor R 0 (Å) A 0 (M) a Ref.
Phe Tyr 11.5–13.5 0.29–0.18 82–84
Tyr Tyr 9–16 0.61–0.11 12, 13
Tyr Trp 9–18 0.61–0.08 12, 85–88
Trp Trp 4–16 7.0–0.11 12, 89
a The critical concentration (A0 ) in moles/liter can be calculated from A 0
= 447/R 0 3, where R 0 is in Å. See Chapter 13.
cous solvent, where the Stokes shift was smaller, the R 0
value for tryptophan homotransfer 12 was found to be as
large as 16 Å. Hence trp-to-trp transfer can be expected in
proteins, particularly if some of the residues display a blueshifted
emission.
16.4.1. Tyrosine to Tryptophan Energy Transfer in
Interferon-γ
The most commonly observed resonance energy transfer in
proteins is from tyrosine to tryptophan. This is because
most proteins contain both of these amino acids, and both
are readily excited at 275 nm. One example of tyr-to-trp
transfer is human interferon-γ, whose emission spectrum
depends on the extent of self-association. Interferon-γ is
produced by activated lymphocytes and displays antiviral
and immunoregulator activity. Its activity depends on the
extent of association to dimers. The intrinsic fluorescence
of interferon-γ was used to study its dissociation into
monomers.
Interferon-γ is usually a dimer of two identical 17, kDa
polypeptides, each containing one tryptophan residue at
position 36 and four tyrosine residues (Figure 16.17). 90 The
emission spectrum of the interferon-γ dimer (Figure 16.18,
top) displays emission from both tyrosine and tryptophan
when excited at 270 nm. Only tryptophan emission is seen
for 295-nm excitation. To compare the relative intensities of
tyrosine and tryptophan the spectra were normalized at long
emission wavelengths where only tryptophan emits, followed
by subtraction of the spectrum with 295-nm excitation
from the spectrum with 270-nm excitation. This difference
spectrum is seen to be that expected for tyrosine.
When incubated under the appropriate conditions, interferon-γ
dissociates into monomers. The relative intensity of
the tyrosine increases in the monomeric state (Figure 16.18,
lower panel). In the monomeric state the relative tyrosine
intensity is about half that of tryptophan. In the dimer the