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

458 Chapter 8: Analyzing Cells, Molecules, and Systems
the antibody, the partner precipitates as well and can be identified by mass spectrometry.
This method is useful for identifying proteins that are part of a complex
inside cells, including those that interact only transiently—for example, when
extracellular signal molecules stimulate cells (discussed in Chapter 15).
In addition to capturing protein complexes on columns or in test tubes,
researchers are developing high-density protein arrays to investigate protein
interactions. These arrays, which contain thousands of different proteins or antibodies
spotted onto glass slides or immobilized in tiny wells, allow one to examine
the biochemical activities and binding profiles of a large number of proteins
at once. For example, if one incubates a fluorescently labeled protein with arrays
containing thousands of immobilized proteins, the spots that remain fluorescent
after extensive washing each contain a protein that specifically binds the labeled
protein.
Optical Methods Can Monitor Protein Interactions
Once two proteins—or a protein and a small molecule—are known to associate,
it becomes important to characterize their interaction in more detail. Proteins
can associate with each other more or less permanently (like the subunits of RNA
polymerase or the proteasome), or engage in transient encounters that may last
only a few milliseconds (like a protein kinase and its substrate). To understand
how a protein functions inside a cell, we need to determine how tightly it binds to
other proteins, how rapidly it dissociates from them, and how covalent modifications,
small molecules, or other proteins influence these interactions.
As we discussed in Chapter 3 (see Figure 3–44), the extent to which two proteins
interact is determined by the rates at which they associate and dissociate.
These rates depend, respectively, on the association rate constant (k on ) and dissociation
rate constant (k off ). The kinetic rate constant k off is a particularly useful
number because it provides valuable information about how long two proteins
remain bound to one another. The ratio of the two kinetic constants (k on /k off )
yields another very useful number called the equilibrium constant (K, also known
as K eq or K a ), the inverse of which is the more commonly used dissociation constant
K d . The equilibrium constant is useful as a general indicator of the affinity
of the interaction, and it can be used to estimate the amount of bound complex at
different concentrations of the two protein partners—thereby providing insights
into the importance of the interaction at the protein concentrations found inside
the cell.
A wide range of methods can be used to determine binding constants for a
two-protein complex. In a simple equilibrium binding experiment, two proteins
are mixed at a range of concentrations, allowed to reach equilibrium, and the
amount of bound complex is measured; half of the protein complex will be bound
at a concentration that is equal to K d . Equilibrium experiments often involve the
use of radioactive or fluorescent tags on one of the protein partners, coupled with
biochemical or optical methods for measuring the amount of bound protein. In
a more complex kinetic binding experiment, the kinetic rate constants are determined
using rapid methods that allow real-time measurement of the formation
of a bound complex over time (to determine k on ) or the dissociation of a bound
complex over time (to determine k off ).
Optical techniques provide particularly rapid, convenient, and accurate binding
measurements, and in some cases the proteins do not even need to be labeled.
Certain amino acids (tryptophan, for example) exhibit weak fluorescence that can
be detected with sensitive fluorimeters. In many cases, the fluorescence intensity,
or the emission spectrum of fluorescent amino acids located in a protein–protein
interface, will change when two proteins associate. When this change can
be detected by fluorimetry, it provides a simple and sensitive measure of protein
binding that is useful in both equilibrium and kinetic binding experiments. A
related but more widely useful optical binding technique is based on fluorescence
anisotropy, a change in the polarized light that is emitted by a fluorescently tagged
protein in the bound and free states (Figure 8–19).