Principles of Fluorescence Spectroscopy

PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 551
Figure 16.38. Stern-Volmer plot for acrylamide quenching of S.
aureus metalloprotease (dotted). Excitation wavelength was 297 nm
and emission was observed at 336 nm. The solid line shows the leastsquares
fit with parameters K 1 = 14.1 M –1 , K 2 = 0.52 M –1 , f 1 = 0.52,
and f 2 = 0.48. Revised from [116].
state. A single-tryptophan residue can display a multi-exponential
decay, and the decays can depend on wavelength.
Hence, there is no a priori reason to assume the residue is
quenched with the same quenching constants at all emission
wavelengths.
16.7. ASSOCIATION REACTION OF PROTEINS
An important use of intrinsic protein fluorescence is to
study binding interactions of proteins. 118–120 Such measurements
take advantage of the high sensitivity of fluorescence
Figure 16.39. Resolution of the two tryptophan emission spectra from
S. aureus metalloprotease. ", component with K 1 = 0.52 M –1 ; ∆, component
with K 2 = 14.1 M –1 ; !, steady-state spectrum. Revised from
[116].
and the ability to perform measurements on dilute protein
solutions. Detection of protein association reactions by fluorescence
is made possible by the high sensitivity of tryptophan
to its local environment. A change in exposure to
solvent or a change in proximity to a quenching group can
often change the emission maximum or quantum yield of
tryptophan residues.
16.7.1. Binding of Calmodulin to a Target Protein
Intrinsic protein fluorescence was used to study binding of
calmodulin to a target protein. The target protein was a peptide
fragment from a glutamate receptor. Glutamate is the
dominant neurotransmitter in the human brain. The neuronal
receptor peptide (NRP) contains a single-tryptophan
residue. Calmodulin does not contain tryptophan, which
allowed emission from the NRP to be observed selectively
using 297-nm excitation. The intrinsic tryptophan emission
of the NRP was used to study its binding to calmodulin in
the absence and presence of calcium. It is frequently
assumed that calmodulin does not interact with target proteins
until it binds calcium. However, the absence of any
interaction in the absence of calcium could result in a slow
cellular response to calcium because of the time needed for
diffusive encounters to occur. Pre-association of target proteins
with calmodulin could increase the response speed to
calcium transients.
Emission spectra of the NRP are shown in Figure
16.40. Upon addition of calcium and calmodulin to the
NRP there was an increase in intensity and a blue shift of its
tryptophan emission, 120 showing that calcium-saturated calmodulin
binds to the NRP (lower left). Surprisingly, there
was also an increase in intensity and blue shift of the NRP
emission with calmodulin in the absence of calcium (upper
left), showing that binding occurs without calcium. This
result indicated that calmodulin pre-associates with the
receptor, presumably to provide a more rapid response to
calcium transients.
If calmodulin binds to the receptor without calcium
then how does calcium trigger the receptor? This question
was partially answered by studies of the N and C domains
of calmodulin. The C domain alone was found to interact
with the NRP in the absence of calcium (Figure 16.40,
upper right). Interaction between the C domain and calmodulin
was increased by calcium. The N domain was found to
interact with the NRP only in the presence of calcium (middle
panels). These results suggest that the C domain is
responsible for pre-association and the N domain for signal-