Principles of Fluorescence Spectroscopy

PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 303
Figure 8.39. Three-dimensional structure of the E. coli tet repressor.
The position of a bound DNA oligomer is shown for Problem 8.10.
Figure courtesy of Dr. Oliver Scholz from the Friedrich-Alexander
University of Erlangen-Neuremberg, Germany.
tetracycline. This protein is a symmetrical dimer that contains
two tryptophan residues in each subunit at positions
43 and 75 (Figure 8.39). W43 is thought to be an exposed
residue, and W75 is thought to be buried in the protein
matrix. 107 Earlier studies of single-tryptophan mutants of
the tet repressor confirmed the accessibility of W43 to
iodide and the shielding of W75 from iodide quenching. 108
Hence, this protein provided an ideal model protein to
attempt quenching resolution of the individual emission
spectra of two tryptophan residues in a protein.
Iodide Stern-Volmer plots for the tet repressor were
measured for various emission wavelengths 107 (Figure
8.40). A larger amount of quenching was observed at longer
wavelengths. When analyzed in terms of two components,
one of these components was found to be almost inaccessi-
Figure 8.40. Iodide Stern-Volmer plots for the wild type tet repressor.
The solution contained 1 mM sodium thiosulfate to prevent formation
of I 2 . From [107].
Figure 8.41. Fluorescence quenching-resolved spectra of wild-type
tet repressor using potassium iodide as the quencher. The solid line is
the unquenched emission spectrum. Top: wavelength-dependent values
of K 1 . From [107].
ble to iodide. At 324 nm the recovered values for the more
accessible fractions are f 1 = 0.34 and K 1 = 16.2 M –1 . For the
inaccessible fraction the values are f 2 = 0.66 and K 2 = 0. The
wavelength-dependent data were used to calculate the individual
spectra (Figure 8.41). The blue-shifted emission with
a maximum of 324 nm corresponds to the inaccessible fraction,
and the red-shifted spectrum at 349 nm is the fraction
accessible to iodide quenching. These emission spectra are
assigned to W75 and W43, respectively. The results in Figure
8.41 (top) illustrate one difficulty often encountered in
determination of quenching-resolved spectra. The quenching
constant for a single species can be dependent on emission
wavelength. In this case the quenching constant of the
accessible tryptophan changed about twofold across its
emission spectrum. When this occurs the values of K i (λ)
cannot be treated as global parameters.
Single-tryptophan mutants of the tet repressor were
used to study the accessibility of each trp residue to iodide
quenching (Figure 8.42). Little if any quenching was
observed for the protein containing only tryptophan 75, and
tryptophan 43 was readily quenched by iodide. 109 Iodide