Principles of Fluorescence Spectroscopy

PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 539
Figure 16.14. α-Carbon backbone of wild-type azurin from
Pseudomonas aeruginosa. The environment around the single-tryptophan
residue (W48) was varied by mutating isoleucine (I7) or phenylalanine
(F110).
Site-directed mutagenesis of azurin was used to change
amino acids located near the tryptophan residue. A single
amino acid substitution was found to eliminate the structured
emission spectrum of tryptophan. In a mutant protein
the nonpolar amino acid isoleucine (I) at position 7 was
replaced with serine (S). This mutant protein is referred to
as I7S. The amino acid serine contains a hydroxyl group,
which might be expected to form a hydrogen bond to indole
and thus mimic ethanol in cyclohexane (Figure 16.5). This
single amino acid substitution resulted in a complete loss of
the structured emission (Figure 16.15). The emission maximum
is still rather blue shifted (λ max = 313 nm), reflecting
the predominantly nonpolar character of the indole environment.
The emission of tryptophan 48 was also found to be
sensitive to substitution of the phenylalanine residue at
position 110 by serine (F110S, Figure 16.15). These studies
demonstrate that just a single hydrogen bond can eliminate
the structured emission of indole, and that the emission
spectra of indole are sensitive to small changes in the local
environment.
Figure 16.15. Corrected steady-state fluorescence spectra of
holoazurin Pae: WT (solid), I7S (dotted), F110S (dashed). Data were
collected at 298°K in 20 mM Hepes buffer at pH 8.0. The excitation
wavelength was 285 nm. Reprinted with permission from [79].
Copyright © 1987, American Chemical Society.
16.3.2. Emission Spectra of Azurins with One or
Two Tryptophan Residues
The azurins also provide examples of single-tryptophan
proteins with the tryptophan residues located in different
regions of the protein. 79 Azurins isolated from different
microorganisms have somewhat different sequences.
Azurin Pae has a single buried tryptophan residue at position
48. Azurin Afe has a single tryptophan on the surface
at position 118, and azurin Ade has tryptophan residues at
both positions 48 and 118. Each of these azurins displays
distinct emission spectra (Figure 16.16). The emission of
the buried residue W48 is blue shifted, and the emission of
the exposed residue W118 displays a red-shifted featureless
spectrum. The emission spectrum of azurin Ade with both
residues is wider and shows emission from each type of
tryptophan residue. Hence, a wide range of environments
can exist within a single protein.
16.4. ENERGY TRANSFER AND INTRINSIC
PROTEIN FLUORESCENCE
Resonance energy transfer can occur between the aromatic
amino acids in proteins. Transfer is likely to occur because
of spectral overlap of the absorption and emission spectra
of phe, trp, and trp (Figure 16.1). The local concentrations
of these residues in proteins can be quite large. Consider a
typical globular protein with a molecular weight near