Principles of Fluorescence Spectroscopy

PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 373
Figure 10.21. Anisotropy of the fluorescein (F)-labeled repressor
DNA sequence upon titration with the Trp repressor (TR). Revised
and reprinted with permission from [52]. Copyright © 1993,
American Chemical Society.
contributes more strongly to the measured anisotropy.
Under these conditions the fraction bound is given by
f B
r r F
(r r F) R(r B r)
(10.51)
where R = f B /f F is the ratio of intensities of the bound and
free forms. Note that in eq. 10.51 the value of f B represents
the fractional concentration and not the fractional intensity.
The fractional concentrations can be used to calculate the
dissociation constant for the reaction.
10.7.2. Binding of the Trp Repressor to DNA
Studies of tryptophan repressor protein binding to DNA 52
are other examples of anisotropy measurements. In the
presence of tryptophan this protein binds to several regions
of the E. coli genome, one of which controls tryptophan
synthesis. Binding to this region of the genome suppresses
the transcription of genes for proteins that are in the tryptophan
synthesis pathway. The trp repressor binds to the DNA
sequence shown in Figure 10.21. This double-stranded
sequence was labeled with fluorescein on one of its 5' ends.
Upon addition of the repressor protein the fluorescein
anisotropy increases due to the decreased rotational rate of
the DNA 25-mer when bound to the repressor. The concentration
of repressor needed for binding was strongly
dependent on the concentration of tryptophan in solution.
The binding to DNA was much stronger in the presence of
tryptophan, which can be seen from the anisotropy increasing
at lower TR concentrations. This is consistent with the
known function of the repressor, which is to turn off the
genes responsible for tryptophan synthesis when tryptophan
levels are adequate.
The titration curves shown in Figure 10.21 can be
understood in terms of a model of Trp repressor binding to
DNA (Figure 10.22). Binding of tryptophan to the repressor
increases the repressor affinity for DNA. This model also
explains another feature of the titration curves, which is the
further increase in anisotropy at higher repressor concentrations
(Figure 10.21). Apparently, the DNA 25-mer can bind
more than a single repressor dimer, and this additional binding
occurs at higher repressor concentrations.
10.7.3. Helicase-Catalyzed DNA Unwinding
Anisotropy measurements are rather simple and can be
made rapidly. This allows anisotropies to be used to study
reaction kinetics occurring in fractions of a second. 53–55 One
example is unwinding of double-helical DNA by helicase. 56
Helicases are found in all species from humans to bacteria.
Unwinding of DNA is necessary for DNA replication. Helicases
move along DNA in a single direction and destabilize
the DNA base pairs using energy derived from ATP. The
helicases typically prefer to act on oligomers that have a
single-stranded region.
Figure 10.22. Binding of the Trp repressor to DNA. Revised and reprinted with permission from from [52]. Copyright © 1993,
American Chemical Society.