Principles of Fluorescence Spectroscopy

372 FLUORESCENCE ANISOTROPY
of the probe and/or domains of the F ab fragment. Hence, the
y-intercepts are larger. Perrin plots that depend differently
on temperature or viscosity should not be regarded as incorrect,
but rather as reflecting the temperature-dependent
dynamics of the protein.
10.7. BIOCHEMICAL APPLICATIONS OF
STEADY-STATE ANISOTROPIES
Anisotropy measurements are ideally suited for measuring
the association of proteins with other macromolecules. This
is because the anisotropy almost always changes in
response to a change in correlation time. Also, the experiments
are simplified by the independence of the anisotropy
from the overall protein concentration.
10.7.1. Peptide Binding to Calmodulin
The use of anisotropy to study protein binding is illustrated
by calmodulin. This protein activates a number of intracellular
enzymes in response to calcium. One example is
myosin light-chain kinase (MLCK). 49 The amino-acid
(RS20) sequence that binds to calmodulin contains a single
tryptophan residue (Figure 10.19). Since calmodulin contains
only tyrosine, the peptide (RS20) can be selectively
observed by excitation at 295 nm (Chapter 16). Upon addition
of calmodulin the emission intensity at RS20 increases
and the emission shifts to shorter wavelengths. These
changes indicate a shielded environment for the tryptophan
Figure 10.19. Emission spectra of the myosin light-chain kinase
(MLCK) peptide RS20 in solution (solid) and bound to calmodulin in
the presence of calcium (dashed). Excitation at 295 nm. Revised and
reprinted with permission from [49]. Copyright © 1986, American
Chemical Society.
Figure 10.20. Titration of the MLCK peptide RS20 with calmodulin.
Revised and reprinted with permission from [49]. Copyright © 1986,
American Chemical Society.
residue in the complex. The anisotropy of RS20 increases
dramatically on addition of calmodulin (Figure 10.20).
These data can be used to determine that the stoichiometry
of binding is 1:1. The sharp nature of the transition at 10 –8
M calmodulin implies that the binding constant is less than
10 –9 M.
How can such data be used to calculate the fraction of
the peptide that is free in solution (f F ) and the fraction
bound to calmodulin (f B )? The additivity law for
anisotropies (eq. 10.6) is appropriate when f i are the fractional
intensities, not the fractional populations. Near 340
nm there is no change in intensity upon binding. If the
polarized intensities are measured at this isoemissive point,
the fraction of RS20 bound can be calculated using 50–51
f B r r F
r B r F
(10.50)
where r is the measured anisotropy, and r F and r B are the
anisotropies of the free and bound peptides, respectively.
The value of r F is obtained by measuring the anisotropy of
the free fluorophores, in this case the RS20 peptide prior to
addition of calmodulin. The value of r B is typically obtained
from the limiting value observed when binding is thought to
be complete. At different observation wavelengths the
measured anisotropy can be weighted toward the free or
bound forms. For instance, at 320 nm the bound peptide