Protein Structure and

Function
(3rd half)

How proteins are studied

Understanding how a particular protein functions
requires detailed structural and biochemical
analysesboth of which require large amounts of
pure protein. For many years, proteins had to be
purified directly from the source: the tissues in
which they are most plentiful. That approach was
inconvenient. 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. Often these cells have even been
tricked into making large quantities of a given
protein using the genetic engineering techniques.

Cells can be grown in a culture dish

Given the appropriate surroundings, most plant and
animal cells will live, proliferate, and even express
specialized properties in a tissue-culture dish.
Experiments performed using cultured cells are
sometimes said to be carried out in vitro (literally, in
glass) to contrast them with experiments on intact
organisms, which are said to be carried out in vivo
(literally, in the living). Because these phenomena
occur in culture, in a controlled environment, they are
accessible to study in ways that are often not possible in
intact tissues. For example, cultured cells can be exposed
to hormones or growth factors, and the effects that these
molecules have on the shape or behavior of the cellsor
on the proteins they produce in responsecan be easily
explored.

Purification techniques allow homogeneous protein

preparations to be obtained from cell homogenates
The first step in any purification procedure is to break open the
cells to release their contents; the resulting slurry is called a cell
homogenate. This physical disruption is followed by an initial
fractionation procedure to separate out the class of molecules of
interestfor example, all the soluble proteins in the cell.
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
separate the individual components of a complex mixture into
different portions, or fractions. After each such step, one uses
some sort of assayfor example, a test for the proteins activity
to determine which fractions contain the protein of interest.
Such fractions are then subjected to additional chromatography
steps until the desired protein is obtained in pure form.

 The most popular forms of protein chromatography

separate polypeptides on the basis of their size, their
charge, or their ability to bind to a particular chemical
group. If antibodies that recognize a particular protein
are available, they can be used to help extract that
protein from a mixture.
Proteins can also be separated by electrophoresis. In
this technique, a mixture of proteins is loaded onto a
polymer gel and subjected to an electric field; the
polypeptides will then migrate through the gel at
different speeds depending on their size and net
charge. If too many proteins are present in the sample,
or if the proteins are very similar in their migration rate,
they can be resolved further using two dimensional gel
electrophoresis. These electrophoretic approaches yield
a number of bands or spots that can be visualized by
staining, each one containing a different protein.

 Once a protein has been obtained in

pure form, it can be used in biochemical
assays to study the details of its
activity, and it can be subjected to
techniques that reveal its amino acid
sequence and precise threedimensional structure

Large amounts of almost any protein can be produced

by
genetic engineering techniques

Companies now use bacteria, yeast, or

cultured mammalian cells to mass produce
all sorts of proteins that are used
therapeutically, such as insulin, human
growth hormone etc. Using the same sorts of
genetic engineering techniques, scientists
can also design proteins that perform novel
tasks: metabolizing toxic wastes,
synthesizing life-saving drugs, or operating
under conditions that would destroy most
biological catalysts.

Automated studies of protein structure and function

are
increasing the pace of discovery

Biochemists have made enormous progress in

understanding the structure and function of proteins
over the past 150 years but many future advances
may come from proteomics, the large scale study of
cellular proteins in which the activities or structures of
hundredseven thousandsof proteins are analyzed
by highly sensitive, automated techniques. Largescale analyses of protein structures are already under
way. Techniques that have been miniaturized and
automated allow researchers to rapidly clone genes,
produce proteins, grow crystals, and collect X-ray
diffraction data for hundreds of proteins at a time.

 Through X-ray crystallography and

nuclear magnetic resonance (NMR)
spectroscopy, we now know the
three-dimensional shapes of more
than 20,000 proteins.

Probing protein structure

Scientists use two main methods to map the location of
atoms in a protein. The first involves the use of X-rays.
A second method, called nuclear magnetic resonance (NMR)
spectroscopy, takes advantage of the fact that the nuclei of
many atoms are intrinsically magnetic. When exposed to a
larger magnet, these nuclei act like tiny bar magnets and
align themselves with the magnetic field. If they are then
excited with a blast of radio waves, the nuclei will wobble
around their magnetic axes, and, as they relax back into
position, they will give off a signal that can be used to
reveal their relative positions in a protein. But before these,
large amounts of the protein have to be isolated in a pure
form. You must also know its amino acid sequence, so that
the data you collect about the way its atoms are arranged
can be interpreted.

Fingerprints
Before a protein is sequenced, it is
generally broken into smaller pieces with
a selective protease. The enzyme trypsin,
for example, cleaves polypeptide chains
on the carboxyl side of a lysine or an
arginine. So if a protein has nine lysines
and seven arginines, digestion with
trypsin should cut it into 17 peptide
fragments. The identities of the amino
acids in each fragment can then be
determined chemically.

 Another, faster way to determine amino acid

sequencesespecially when large numbers of
proteins are being separated and identified at the
same timeis to use a method called mass
spectrometry. This technique determines the
exact mass of every peptide fragment. The peptides
from a tryptic digest are dried onto a metal plate.
The sample is then blasted with a laser, which heats
the peptides, causing them to become electrically
charged (ionized) and ejected from the plate in the
form of a gas. Accelerated by a powerful electric
field, the peptide ions then fly toward a detector,
and the time it takes them to get there is related to
their mass and their charge. (The larger the peptide
is, the more slowly it moves, while the more highly
charged it is, the faster it moves.)

X-rays
Once you have the amino acid sequence of a protein
then to determine a proteins structure using X-ray
crystallography, you first need to coax the protein into
forming crystals: large, highly ordered arrays of the
pure protein in which every molecule has the same
conformation and is perfectly aligned with its neighbors.
When a narrow beam of X-rays is directed at a protein
crystal, the atoms in the protein molecules scatter the
incoming X-rays. These scattered waves either reinforce
or cancel one another, producing a complex diffraction
pattern that is collected by electronic detectors.
Computers are used to interpret them and transform
them by complex mathematical calculations into maps
of the relative spatial positions of atoms.

Magnets

The trouble with X-ray crystallography is that you need

crystals. And not all proteins like to form such orderly
assemblies. If the protein is smallsay, 50,000 daltons
or lessyou can determine its structure by NMR
spectroscopy. In this technique, a concentrated solution
of pure protein is placed in a strong magnetic field and
then bombarded with radio waves of different
frequencies. Its hydrogen nuclei, in particular, will
generate an NMR signal that can be used to determine
the distances between the atoms in different parts of
the protein. This information is then used to build a
model of how these atoms are arranged in space. If the
protein is larger than 50,000 daltons, you can try to
break it up into its constituent functional domains and
analyze each domain by NMR.

Cell breakage and initial

fractionation of cell extracts
The first step in the purification of most proteins is to
disrupt tissues and cells in a controlled fashion. Using
gentle mechanical procedures, called homogenization, the
plasma membranes of cells can be ruptured so that the cell
contents are released. Four commonly used procedures are:
(1)Break cells with high-frequency sound.
(2)Use a mild detergent to make holes in the plasma
membrane.
(3) Force cells through a small hole using high pressure.
(4) Shear cells between a close-fitting rotating plunger and
the thick walls of a glass vessel.
When carefully conducted, homogenization leaves most of
the membrane-enclosed organelles intact.

THE CENTRIFUGE
Centrifugation is the most widely used procedure to
separate a homogenate into different parts, or fractions.
The homogenate is placed in test tubes and rotated at
high speed in a centrifuge or ultracentrifuge. Such speeds
require centrifuge chambers to be refrigerated and
evacuated so that friction does not heat up the
homogenate.
DIFFERENTIAL CENTRIFUGATION
Centrifugation separates cell components on the basis of
size and density. The larger and denser components
experience the greatest centrifugal force and move most
rapidly. They sediment to form a pellet at the bottom of
the tube, while smaller, less dense components remain in
suspension above, a portion called the supernatant.

 VELOCITY SEDIMENTATION
Subcellular components sediment at different rates
according to their size after being carefully layered
over a dilute salt solution and then centrifuged
through it. In order to stabilize the sedimenting
components against convective mixing in the tube,
the solution contains a continuous shallow gradient
of sucrose that increases in concentration toward
the bottom of the tube. This is typically 520%
sucrose. When sedimented through such a dilute
sucrose gradient, different cell components
separate into distinct bands that can be collected
individually. After an appropriate centrifugation time
the bands may be collected, most simply by
puncturing the plastic centrifuge tube and collecting
drops from the bottom

EQUILIBRIUM
SEDIMENTATION
The ultracentrifuge
can also be used to separate cell
components on the basis of their buoyant density,
independently of their size or shape. The sample is
usually either layered on top of, or dispersed within, a
steep density gradient that contains a very high
concentration of sucrose or cesium chloride. Each
subcellular component will move up or down when
centrifuged until it reaches a position where its
density matches its surroundings and then will move
no further. A series of distinct bands will eventually
be produced, with those nearest the bottom of the
tube containing the components of highest buoyant
density. The method is also called density gradient
centrifugation.

Protein separation by
chromatography

THREE KINDS OF CHROMATOGRAPHY

Although the material used to form the matrix for column

chromatography varies, it is usually packed in the column in
the form of small beads. A typical protein purification strategy
might employ in turn each of the three kinds of matrix
described below, with a final protein purification of up to
10,000-fold. Proteins are often fractionated by column
chromatography. A mixture of proteins in solution is applied to
the top of a cylindrical column filled with a permeable solid
matrix immersed in solvent. A large amount of solvent is then
pumped through the column. Because different proteins are
retarded to different extents by their interaction with the
matrix, they can be collected separately as they flow out from
the bottom. According to the choice of matrix, proteins can be
separated according to their charge, hydrophobicity, size, or
ability to bind to particular chemical groups.

(A) ION-EXCHANGE
CHROMATOGRAPHY
Ion-exchange columns are packed with
small beads carrying either positive or
negative charges that retard proteins of
the opposite charge. The association
between a protein and the matrix
depends on the pH and ionic strength of
the solution passing down the column.
These can be varied in a controlled way
to achieve an effective separation.

(B) GEL-FILTRATION
CHROMATOGRAPHY
Gel-filtration columns separate proteins
according to their size. The matrix
consists of tiny porous beads. Protein
molecules that are small enough to enter
the holes in the beads are delayed and
travel more slowly through the column.
Proteins that cannot enter the beads are
washed out of the column first. Such
columns also allow an estimate of protein
size.

(C) AFFINITY
CHROMATOGRAPHY
Affinity columns contain a matrix
covalently coupled to a molecule that
interacts specifically with the protein of
interest (e.g., an antibody, or an enzyme
substrate). Proteins that bind specifically
to such a column can subsequently be
released by a pH change or by
concentrated salt solutions, and they
emerge highly purified

Protein separation by
electrophoresis
GEL ELECTROPHORESIS
When an electric field is applied to a solution containing
protein molecules, the molecules will migrate in a direction
and at a speed that reflects their size and net charge. This
forms the basis of the technique called electrophoresis.
ISOELECTRIC FOCUSING
For any protein there is a characteristic pH, called the
isoelectric point, at which the protein has no net charge
and therefore will not move in an electric field. In
isoelectric focusing, proteins are electrophoresed in a
narrow tube of polyacrylamide gel in which a pH gradient
is established by a mixture of special buffers. Each protein
moves to a point in the gradient that corresponds to its
isoelectric point and stays there.

SDS polyacrylamide-gel
electrophoresis (SDS-PAGE)
Individual polypeptide chains form a
complex with negatively charged molecules
of sodium dodecyl sulfate (SDS) and
therefore migrate as a negatively charged
SDSprotein complex through a slab of
porous polyacrylamide gel. A reducing agent
(mercaptoethanol) is usually added to break
any SS linkages in or between proteins.
Under these conditions, proteins migrate at
a rate that reflects their molecular weight.

TWO-DIMENSIONAL POLYACRYLAMIDEGEL ELECTROPHORESIS

Complex mixtures of proteins cannot be resolved
well on one-dimensional gels, but two-dimensional
gel electrophoresis, combining two different
separation methods, can be used to resolve more
than 1000 proteins in a two-dimensional protein
map. In the first step, native proteins are
separated in a narrow gel on the basis of their
intrinsic charge using isoelectric focusing. In the
second step, this gel is placed on top of a gel slab,
and the proteins are subjected to SDS-PAGE in a
direction perpendicular to that used in the first
step. Each protein migrates to form a discrete spot.