Principles of Fluorescence Spectroscopy

PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 573
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PROBLEMS
P16.1. Determination of Protein Association and Unfolding
by Fluorescence: Suppose you have a protein that consists
of a single subunit with a molecular weight of
25,000 daltons. The protein contains a single-tryptophan
residue near the central core of the protein and
several tyrosine residues. The protein also contains a
single reactive sulfhydryl residue on the surface.
A. Assume that the monomeric protein can be
unfolded by the addition of denaturant. Explain
how the fluorescence spectral properties of the
unmodified protein could be used to follow the
unfolding process.
B. Describe the use of collisional quenchers to
probe the accessibility of the tryptophan residue
to the solvent.
C. Assume that the unmodified protein self-associates
with another subunit to form a dimer. How
could fluorescence spectroscopy be used to follow
the association process?
D. Describe how you would use fluorescence spectroscopy
to measure the distance from the tryptophan
residue to the reactive sulfhydryl group.
Be specific with regard to the experiments that
you would perform and how the data would be
interpreted.
E. Describe how you would use energy transfer to
measure self-association of the protein after the
protein has been modified on the sulfhydryl
groups with dansyl chloride.
P16.2. Detection of Protein Dimerization: Suppose you
have a small protein with a single-tryptophan
residue that displays r 0 = 0.30, and that the protein
associates to a dimer. The correlation times of the
monomer and dimer are θ M = 1.25 and θ D = 2.5 ns,
respectively. Upon dimer formation the lifetime
increases from τ M = 2.5 to τ D = 5.0 ns, and the relative
quantum yield increases twofold. Describe how
you would detect dimer formation using the:
A. steady-state intensity,
B. intensity decay,
C. steady-state anisotropy, or
D. anisotropy decay.
E. What fraction of the emission is due to the
monomers and dimers when 50% of the
monomers have formed dimers? What is the
steady-state anisotropy? What are the intensity
and anisotropy decays?
P16.3. Effect of Excitation Wavelength on Protein Fluorescence:
Section 16.9.3 described kinetic studies of
the refolding of cellular retinoic acid biding protein
I (CRABPI). The wild-type protein contains three
tryptophan residues (Figure 16.57). Figure 16.74
shows emission spectra of the native and denatured
forms of CRABPI for various excitation wavelengths.
The emission spectra of native CRABPI
shift to longer wavelengths with increasing excitation
wavelength. The emission spectra of denatured
CRABPI do not depend on excitation wavelength.
Explain these emission spectra. There may be more
than one explanation.
P16.4. Binding Between Calmodulin and a Protein Kinase
C: Myristoylated protein kinase (MRP) is a kinase
that binds to calmodulin. MRP and CaM both lack
tryptophan with wild-type sequence MRP. Emission
spectra of wild-type and single-tryptophan mutants
of MRP are shown in Figure 16.75. Explain these
spectra.