Essential Cell Biology 5th edition

158 CHAPTER 4 Protein Structure and Function
protein scaffolds
amyloid
product
(A)
RNA scaffolds
(B) (C) (D)
2 µm 1 µm 1 µm
Figure 4−54 Intracellular condensates
can form biochemical subcompartments
in cells. These large aggregates form as a
result of multiple weak binding interactions
between scaffolds and other macromolecules.
When these macromolecule–macromolecule
interactions become sufficiently strong, a
“phase separation” occurs. This creates two
distinct aqueous compartments, in one of
which the interacting molecules are densely
aggregated. Such intracellular condensates
concentrate a select set of macromolecules,
thereby producing regions with a special
biochemistry without the use of an
encapsulating membrane.
(A) Schematic illustration of a phaseseparated
intracellular condensate. These
condensates can create a factory that
catalyzes the formation of a specific type of
product, or they can serve to store important
entities, such as specific mRNA molecules,
for later use. As shown, reversible amyloid
structures often help to create these
aggregates. These β-sheet structures form
between regions of unstructured amino acid
sequence within the larger protein scaffolds.
(B–D) Three examples that illustrate how
intracellular condensates (colorized regions)
are thought to be used by cells. (B) Inside the
interphase nucleus, the nucleolus is a large
factory that produces ribosomes. In addition,
many scattered RNA production factories
concentrate the protein machines that
transcribe the genome. (C) In the cytoplasm,
a matrix forms the centrosome that nucleates
the assembly of microtubules. (D) In a patch
underlying the plasma membrane at the
synapse where communicating nerve cells
touch, multiple interacting scaffolds produce
large protein assemblies; these create a local
biochemistry that makes possible memory
formation and storage in the nerve cell
network. (B, courtesy of E.G. Jordan and
J. McGovern; C, from M. McGill,
D.P. Highfield, T.M. Monahan, and
B.R. Brinkley, J. Ultrastruct. Res. 57:43–53,
1976. With permission from Elsevier;
D, courtesy of Cedric Raine.)
“hydrogel” that pulls other molecules into the condensate (Figure 4−54).
Amyloid-forming proteins thus have functional roles in cells. But for a
handful of these amyloid-forming proteins, mutation or perturbation can
lead to neurological disease, which is how some of them were initially
discovered.
HOW PROTEINS ARE STUDIED
ECB5 04.54
Understanding how a particular protein functions calls for detailed structural
and biochemical analyses—both of which require large amounts of
pure protein. But isolating a single type of protein from the thousands
of other proteins present in a cell is a formidable task. For many years,
proteins had to be purified directly from the source—the tissues in which
they are most plentiful. That approach was inconvenient, entailing, for
example, early-morning trips to the slaughterhouse. More importantly,
the complexity of intact tissues and organs is a major disadvantage when
trying to purify particular molecules, because a long series of chromatography
steps is generally required. These procedures not only take weeks
to perform, but they also yield only a few milligrams of pure protein.
Nowadays, proteins are more often isolated from cells that are grown in
a laboratory (see, for example, Figure 1−39). Often these cells have been
“tricked” into making large quantities of a given protein using the genetic
engineering techniques discussed in Chapter 10. Such engineered cells
frequently allow large amounts of pure protein to be obtained in only a
few days.
In this section, we outline how proteins are extracted and purified from
cultured cells and other sources. We describe how these proteins are
analyzed to determine their amino acid sequence and their three-dimensional
structure. Finally, we discuss how technical advances are allowing
proteins to be analyzed, cataloged, manipulated, and even designed from
scratch.
Proteins Can Be Purified from Cells or Tissues
Whether starting with a piece of liver or a vat of bacteria, yeast, or animal
cells that have been engineered to produce a protein of interest, the
first step in any purification procedure involves breaking open the cells
to release their contents. The resulting slurry is called a cell homogenate
or extract. This physical disruption is followed by an initial fractionation
procedure to separate out the class of molecules of interest—for example,
all the soluble proteins in the cell (Panel 4−3, pp. 164–165).
With this collection of proteins in hand, the job is then to isolate the
desired protein. The standard approach involves purifying the protein
through a series of chromatography steps, which use different materials
to separate the individual components of a complex mixture into