Molecular Biology of the Cell by Bruce Alberts, Alexander Johnson, Julian Lewis, David Morgan, Martin Raff, Keith Roberts, Peter Walter by by Bruce Alberts, Alexander Johnson, Julian Lewis, David Morg

ANALYZING PROTEINS
459
FAST
TUMBLING
depolarized
emission
280
polarization
filter
free molecule X
SLOW
TUMBLING
polarized
emission
low anisotropy
anisotropy (mP)
200
120
depolarized
excitation
light
polarized
excitation
light
high anisotropy
40
0 10 20 30 40 50
receptor concentration (µM)
(A)
bound
molecule X
receptor
(B)
Figure 8–19 Measurement of binding with fluorescence anisotropy. This method depends on a fluorescently tagged
protein that is illuminated with polarized light at the appropriate wavelength for excitation; a fluorimeter is used to measure the
intensity and polarization of the emitted light. If the fluorescent protein is fixed in position and therefore does not rotate during
the brief period between excitation and emission, then the emitted light will be polarized at the same angle as the excitation
light. This directional effect is called fluorescence anisotropy. Protein molecules in solution rotate or tumble rapidly, however,
so that there is a decrease in the amount of anisotropic MBoC6 fluorescence. m8.202/8.21Larger molecules tumble at a slower rate and therefore
have higher fluorescence anisotropy. (A) To measure the binding between a small molecule and a large receptor protein, the
smaller molecule is labeled with a fluorophore. In the absence of its binding partner, the molecule tumbles rapidly, resulting in
low fluorescence anisotropy (top). When the small molecule binds to its larger partner, however, it tumbles less rapidly, resulting
in an increase in fluorescence anisotropy (bottom). (B) In the equilibrium binding experiment shown here, a small, fluorescent
peptide ligand was present at a low concentration, and the amount of fluorescence anisotropy (in millipolarization units, mP) was
measured after incubation with various concentrations of a larger protein receptor for the ligand. From the hyperbolic curve that
fits the data, it can be seen that 50% binding occurred at about 10 μM, which is equal to the dissociation constant K d for the
binding interaction.
Another optical method for probing protein interactions uses green fluorescent
protein (discussed in detail below) and its derivatives of different colors. In this
application, two proteins of interest are each labeled with a different fluorescent
protein, such that the emission spectrum of one fluorescent protein overlaps the
absorption spectrum of the second. If the two proteins come very close to each
other (within about 1–5 nm), the energy of the absorbed light is transferred from
one fluorescent protein to the other. The energy transfer, called fluorescence resonance
energy transfer (FRET), is determined by illuminating the first fluorescent
protein and measuring emission from the second (see Figure 9–26). When combined
with fluorescence microscopy, this method can be used to characterize
protein–protein interactions at specific locations inside living cells (discussed in
Chapter 9).
Protein Function Can Be Selectively Disrupted With Small
Molecules
Small chemical inhibitors of specific proteins have contributed a great deal to the
development of cell biology. For example, the microtubule inhibitor colchicine
is routinely used to test whether microtubules are required for a given biological
process; it also led to the first purification of tubulin several decades ago. In the
past, these small molecules were usually natural products; that is, they were synthesized
by living creatures. Although natural products have been extraordinarily
useful in science and medicine (see, for example, Table 6–4, p. 352), they act on
a limited number of biological processes. However, the recent development of
methods to synthesize hundreds of thousands of small molecules and to carry out
large-scale automated screens holds the promise of identifying chemical inhibitors
for virtually any biological process. In such approaches, large collections of
small chemical compounds are simultaneously tested, either on living cells or in
cell-free assays. Once an inhibitor is identified, it can be used as a probe to identify,
through affinity chromatography or other means, the protein to which the
inhibitor binds. This general strategy, sometimes called chemical biology, has
successfully identified inhibitors of many proteins that carry out key processes
in cell biology. An inhibitor of a kinesin protein that functions in mitosis, for