Chapter 7 Techniques for
Exploring Proteins
Assay: 化验，分析，测定。
Enzyme activity: 1 unit (U) is defined as the
amount of enzyme which convert 1 micromole
(mmol) of substrate to product(s) in 1 minute
under specified conditions.
Specific activity: enzyme activity expressed per
unit mass of protein present. (units/mg)
Red balls=active enzymes. Other color balls=other
proteins. The total activity is the same for the two
cups but the specific activity is higher for the right
cup.
1. Proteins in native conformation can be purified
(separated) according to their size, solubility,
charge, and binding affinity.
1.1 Proteins can be separated from small
molecules by dialysis through a semipermeable
membranes.
1.1.1 Molecules significantly larger than the
diameter of the membrane pores are retained in
the dialysis bag, whereas small ones diffuse out.
1.2 Proteins different in size can be separated by
gel-filtration (size-exclusion, molecular sieve)
chromatography.
1.2.1 Samples are applied to columns of
porous beads (made of insoluble but highly
hydrated polymers like dextran, agarose, and
polyarylamide)
The stationary phase is composed of a porous
matrix and absorbed immobile solvent.
The mobile phase is the flowing solvent
consisted of buffers and salts. (fig.)
1.2.2 Larger protein molecules flow more
rapidly through the column and emerge first
because they cannot enter the internal volume of
the beads.
1.2.3 Proteins smaller than the diameter of
the pores on the beads will enter the labyrinthian
path of the beads, and hence, are slowed in
mobility. (Manufacturer controls the properties,
such as pore size distribution, of the beads.)
1.2.4 Gel-filtration usually has low
resolution.
1.3 The solubility of most proteins is lowered at
high salt concentrations.
1.3.1 This effect is called salting out.
1.3.2 The dependence of solubility on salt
concentration differs from one protein to
another, hence salting out can be used to
fractionate proteins.
1.3.3 Ammonium sulfate precipitation is
often used in fractionating proteins (also to
concentrate proteins).
1.3.4 The mechanism of salting out is not
well understood. (Dehydration is a possible
cause).
1.4 Proteins can be separated on the basis of their
net charge by ion exchange chromatography
1.4.1 Proteins have different net charge at a
given pH due to their differences in pI values (as
for amino acids).
1.4.2 Proteins bind to charged resins with
different affinity, thus being able to be released
(eluted) at different salt concentrations or pH
values (using buffers with a gradient of salt or pH
is run through the column).
1.4.3 The commonly used anion exchange resin is
diethylaminoethyl-cellulose (DEAE-cellulose) and
cation exchange resin is carboxymethyl-cellulose
(CM-cellulose).
1.4.4 Ion exchange chromatography may have
very high resolution (specific binding) and is
routinely used in protein purification.
1.4.5 New view: surface charge distribution is
more crucial than the net charge (pI). pH mapping
and a variety of resins (columns with different
properties) should be tried.
1.5 Proteins can be effectively purified by affinity
chromatography.
1.5.1 This technique makes use of the binding
capacity of many proteins for specific ligands:
chemical groups or (attached) molecules (e.g.,
between substrates and enzymes, antigens and
antibodies, etc.)
1.5.2 The specific ligands are usually
covalently cross-linked to insoluble beads.
1.5.3 Specific ligand-binding proteins are
retained on the column (all other nonspecific
proteins are washed away from the column with
low salt buffers) when a mixture of proteins (cell
extract) is applied.
1.5.4 The specifically bound protein is eluted
out under appropriate conditions (high
concentration of ligands or salts).
2. Proteins can be separated and characterized by
gel electrophoresis.
2.1 Electrophoresis refers to the phenomenon
that a molecule with a net charge will move in an
electric field.
2.1.1 The velocity of migration (v) of a
molecule in an electric field depends on the
electric field strength (E), the net charge (z), and
the friction coefficient (f):
v = Ez/f
2.1.2 The electric force Ez, driving the
charged molecule toward the oppositely charged
electrode, is opposed by the viscous drag (resisting
force) fv, arising from the friction between the
moving molecule and the medium.
2.1.3 The frictional coefficient f depends on
both the mass and shape of the migrating molecule
and the viscosity () of the medium (for a sphere of
radius r, f =6r, Stoke’s law).
2.2 Electrophoresis separation is nearly
always carried out in gels (rather than in free
solutions).
2.2.1 The chemically inert polyacrylamide
gel, formed by the polymerization of acrylamide
monomer cross linked by
methylenebisacrylamide, is routinely used.
2.2.2 The pore size of polyacrylamide gel
can be controlled by choosing various
concentrations of acrylamide and
methylenebisacrylamide at the time of
polymerization (less cross-linking agent,
methylenebisacrylamide, larger pore size).
2.2.3 Gels suppress convective currents
produced by small temperature gradients, a
requirement for effective separation. Gels also
prevent diffusion.
2.2.4 Gels serve as molecular sieves that
enhance separation.
2.2.5 Arne Tiselius won the Nobel Prize in
Chemistry in 1948 for inventing the electrophoresis
technique and applying it to protein studies.
2.3 SDS-polyacrylamide gel electrophoresis
(PAGE) is commonly used for estimation of
protein purity and molecular mass.
2.3.1 The mixture of proteins is first dissolved
in a solution of SDS (sodium dodecyl sulfate), an
anionic detergent that disrupts nearly all
noncovalent interactions in native proteins.
2.3.2 Mercaptoethanol or dithiothreitol is also
added to reduce disulfide bonds.
2.3.3 Anions of SDS bind to peptide main
chains at a ratio of about one SDS for every two
amino acid residues, which gives a complex of SDS
with a denatured protein a large net negative
charge that is roughly proportional to the mass of
the protein.
2.3.4 The native charge of a protein is thus
made insignificant.
2.3.5 The electrophoresis mobility of many
proteins in SDS-PAGE is inversely proportional
to the logarithm of their mass, i.e., smaller
proteins migrate faster than bigger ones.
2.3.6 Proteins separated by SDS-PAGE can be
stained by Coomassie blue (a 0.1 microgram
protein band can be visualized) or silver stain (a
0.002 microgram protein can be visualized).
2.3.7 Protein purity and approximately
molecular mass of proteins separated on SDSPAGE can be estimated.
2.4 Proteins can be separated according to their
isoelectric point (pI).
2.4.1 A pH gradient is first formed in the gel
by electrophoresing a mixture of ampholytes,
which are small multi-charged polymers having
many pI values (a continuous spectrum).
2.4.2 Each protein will move until it reaches a
position in the gel at which the pH is equal to the
pI of the protein.
2.4.3 This method of separating proteins
according to their pI values is called isoelectric
focusing.
2.4.4 Isoelectric focusing can readily resolve
proteins that differ in pI by as little as 0.01,
which means that proteins differing by one net
charge can be separated.
2.4.5 pI values of purified proteins can be
measured by isoelectric focusing.
2.5 Isoelectric focusing can be combined with SDSPAGE to obtain very high-resolution separations.
2.5.1 A single sample is first subject to
isoelectric focusing (separating by pI values) and
then to SDS-PAGE (separating by molecular mass)
in a vertical direction to yield a two-dimensional
pattern of spots.
2.5.2 More than a thousand different proteins
in E.coli can be resolved on a single gel by this
method. (2D gel in proteomics, 3000 proteins and
106 dynamic range.)
3. Ultracentrifugation is valuable for separating
biomolecules and determining their mass under
nondenaturing conditions.
3.1 Protein molecules can be made to sediment
when subjected to very high centrifugal force.
3.1.1 A particle moving in a circle of radius r
at an angular velocity  is subject to a centrifugal
(outward) field equal to  2r.
3.1.2 The centrifugal force Fc on this particle
is equal to the product of its effective mass m’ and
the centrifugal field ( 2r):
Fc = m’  2r = m(1-)  2r
3.1.3 The effective mass m’ is less than the
mass m due to an opposing force generated
from the displaced fluid (i.e., a buoyancy effect,
floating in common phrase).
3.1.4 The buoyancy factor equals to (1-)
where  is the partial specific volume of the
particle (the reciprocal of its density) and  is
the density of the solution.
3.1.5 A particle moves in a centrifugal field at
a constant velocity v (migration velocity or
sedimentation velocity) when the centrifugal force
Fc is equal to the viscous drag vf, where f is the
frictional coefficient of the particle:
v = Fc/f = m(1-)  2r/f
(analogous to the equation for charged particles to
move in an electric field: v = Ez/f)
3.2 Sedimentation coefficient s depends on the
properties of the particle and the solution but is
independent of how fast the sample is spun.
3.2.1 Sedimentation coefficient s is defined as
the velocity divided by the centrifugal field:
s = v/  2r = m(1-) /f
3.2.2 Sedimentation coefficients are usually
expressed in Svedberg units: a svedberg (S) is equal
to 10-13 seconds.
3.2.3 Bigger particles usually have higher s
values.
3.3 The sedimentation velocity of a particle depends
in part on its mass, shape, density of the particle
and solution.
3.3.1 A more massive particle always sediment
more rapidly than does a less massive one of the
same shape and density.
3.3.2 Elongated particles (with higher
frictional coefficient f) sediment more slowly than
do spherical ones of the same mass.
3.3.3 A dense particle moves more rapidly than a
less dense one because the opposing buoyant force
is smaller for the dense particle ( is smaller for
the denser particle).
3.3.4 Particles sink when  < 1, float when
 > 1, do not move when  = 1.
3.4 Proteins of different sedimentation coefficient
can be separated by zonal (band) centrifugation.
3.4.1 A density gradient (for preventing
convective flow) in a centrifuge tube is formed by
mixing different proportions of a low-density
solution (e.g., 5% sucrose) and a high-density one
(e.g., 20% sucrose). (the gradient is pre-formed by
gravity).
3.4.2 A small volume of protein sample is
layered on the top of the density gradient.
3.4.3 Proteins are separated in the density
gradient according to their sedimentation
coefficients: proteins of particular density will tend
to collect in a band at that zone of the centrifuge
tube.
(if isopycnic centrifugation, the protein
density and the density of the medium are exactly
equal, analogous to isoelectric focusing).
3.4.4 The separated bands of proteins can be
fractionated and assayed by making a hole in the
bottom of the tube and collecting drops.
3.5 The mass (molecular weight) of a protein
can be directly determined by sedimentation
equilibrium.
3.5.1 The sample is centrifuged at a relatively
low speed so that sedimentation is counterbalanced
by diffusion.
3.5.2 A smooth gradient of protein
concentration develops during centrifugation.
3.5.3 The dependence of concentration on the
distance from the rotation axis reveals the mass of
the protein molecule.
3.5.4 Native structure of proteins (including
quaternary structure) is preserved during
sedimentation equilibrium centrifugation, thus the
molecular mass of multisubunit proteins can be
determined. An equation involves the diffusion
coefficient, instead of the frictional coefficient.
3.5.5 Sedimentation equilibrium represents
the best way to determine molecular mass of native
proteins.
4. The mass of proteins can be precisely determined
by electrospray mass spectrometry.
4.1 The mass of a protein can be precisely
deduced from the positions of peaks on a mass
spectrum.
4.1.1 A protein sample (highly purified) in
an acidic volatile solvent is sprayed into a mass
spectrometer (The very low volatility of proteins
was a barrier for many years to using mass
spectrometry). (salts and buffers are not volatile
so must be removed).
4.1.2 Solvent surrounding individual droplets
evaporates rapidly in the vacuum chamber,
leaving unfragmented bare protein molecules
carrying multiple positive charges (ionized by the
acidic solvents).
4.1.3 These charged protein molecule are
accelerated by an electric field and then reflected
by a magnetic field.
4.1.4 Proteins are separated according to the
ratio of their mass to their charge (m/z) and a
mass spectrum is thus obtained.
4.1.5 The mass spectrum of a pure protein
shows a set of peaks corresponding to different
numbers of bound protons (adjacent peaks arise
from proteins containing n-1, n, n+1 bound
protons).
4.1.6 The molecular mass of a protein can be
deduced from the positions of the peaks, with an
accuracy of about 0.01% (a 10,000 dalton protein
can be measured to within 1 dalton).
4.1.7 New instruments: MALDI-TOF, ESI
(last year).
5. Proteins can be quatitated and localized by
highly specific antibodies.
5.1 Each antigen evokes a specific set of antibodies,
which will recognize and combine only with that
antigen or closely related molecules (crossreacting).
5.1.1 Antigens represents foreign substances
invading vertebrate animals.
5.1.2 Macromolecules (including proteins,
polysaccharides, nucleic acids) often contain many
eptitopes (antigen determinants), each will illicit
one specific antibody.
5.1.3 Polyclonal antibodies (usually in the
form of antiserum) refers to the mixture of
heterogeneous antibodies that recognize the
different epitopes on a macromolecular antigen
(e.g., a protein).
5.1.4 Monoclonal antibodies refers to the
homogeneous antibodies that recognize one
particular epitope on a macromolecular antigen.
5.1.5 Monoclonal antibodies are generated by
a population of identical (a clone) of cells, and
each such population is descended from a single
hybridoma cell formed by fusing an antibodyproducing spleen cell with a tumor (myeloma) cell
that has the capacity for unlimited proliferation.
5.1.6 Closely related proteins can be
distinguished by antibodies (difference of one
residue on the surface can be detected).
Georges Kohler and Cesar Milstein won the
Nobel Prize in Medicine or Physiology in 1984 for
inventing this method of generating monoclonal
antibodies.
5.2 Antibodies can be used as exquisitely specific
analytic reagents to quantitate the amount of a
protein.
5.2.1 Less than a nanogram (10-9 g) of a
protein can readily be measured by the enzymelinked immunosorbent assay (ELISA): Pregnancy
can be detected within a few days after conception
by immunoassaying urine for the presence of
human chorionic gonadotropin (HCG), a hormone
produced by the placenta.
5.2.2 Very small quantities of a protein of
interest can be detected by Western blotting (find
a needle in a haystack): protein mixture is first
separated on SDS-PAGE and then detected by
antibodies).
5.3 Cells can be stained with labeled antibodies
and examined by microscopy to reveal the
subcellular localization of a protein of interest.
5.3.1 Fluorescence or electron microscopies
can be used. Radiography.
6. Circular dichroism (CD), an expression of
optical acitivity, is a sensitive indicator of the
main-chain conformation of proteins
6.1 Proteins are optically active because they are
dissymmetric (i.e., they cannot be superimposed
onto their mirror images).
6.1.1 Asymmetric centers (the a-carbons of
each residue except Gly, additional asymmetric
centers in Thr and Ile) and the partial double
peptide bonds configurational dissymmetry.
6.1.2 Electronic interactions between
different residues generate conformational
dissymmetry.
6.2 When the left- an right-circularly polarized
light pass through a solution of asymmetric
molecules, they will be absorbed differently.
6.2.1 The measurements of the differential
absorption coefficient () of the two beams at
various wavelength give a CD spectrum.
6.3 The far-ultraviolet CD spectrum of a protein
is sensitive to its main-chain conformation (i.e.,
structural arrangement of peptide bonds).
6.3.1 Various secondary structures show
different CD spectra from 170 to 240 nm.
6.3.2 The CD spectra of a-helices, b
structure motifs and random coils have been
defined by using synthetic polypeptides and
proteins of known structure.
6.3.3 The a-helix makes a dominant
contribution with its negative CD bands at 208 and
222 nm, and its positive band 192 nm. (fig.)
6.3.4 The random coiled structure has a
negative CD band centered at 199 nm.
6.3.5 The CD spectrum of the b sheet
structure has a positive band centered at 198 nm
and a negative band centered at 217 nm.
6.3.6 The percentage of each secondary structure
in a protein can be semiquantitatively predicted
from its CD spectrum (using a set of standard CD
curves generated from proteins of known crystal
structure).
6.3.7 Care must be taken when interpreting
CD spectra (accurate interpretation of a protein’s
CD spectrum is still challenging sometimes.)
7. X-ray crystallography reveals the three
dimensional structure in atomic detail
7.1 Molecules must be precisely oriented and
positioned for analysis.
7.1.1 Crystals of the molecule of interest are
needed (e.g., proteins, nucleic acids, other small
biomolecules).
7.1.2 Slow salting out (often use hang-drop
method) can sometimes lead to the formation of
highly ordered crystals of proteins instead of
amorphous precipitates.
7.1.3 Conditions including pH, precipitant
(e.g., ammonium sulfate, phosphate salts,
polyethylene glycol), buffer, temperature, protein
concentration may affect the crystallization process
of one particular protein.
7.1.4 Generating crystals is often the ratelimiting step in X-ray crystallography and is
mostly still an art.
7.2 X-rays passing through a crystal will be
diffracted (or scattered).
7.2.1 Electrons of an atom scatter X-rays,
with the amplitude of the wave scattered
proportional to its number of electrons (e.g., a
carbon atom scatters six times as strongly as a
hydrogen atom).
7.2.2 The scattered waves recombine
(summed): reinforce each other if they are in
phase (in step); cancel one another if out of phase
(out of step). Each atom contributes to each
scattered beam.
7.2.3 The way in which the scattered waves
recombine depends only on the atomic
arrangement (relative positions to each other).
7.3 X-ray photograph consists of a regular,
three-dimensional array of spots called
reflections.
7.3.1 The intensity of each spot is
measured.
7.3.2 These intensities are the basic
experimental data of an X-ray crystallographic
analysis.
7.4 The image of a protein is reconstructed from
the observed intensities through Fourier transform
(a mathematical treatment).
7.4.1 For each spot, Fourier transform yields a
wave of electron density, whose amplitude is
proportional to the square root of the observed
intensity of the spot.
7.4.2 Each wave has a phase which reflects the
timing of its crests and troughs (peaks and valleys)
relative to those other waves.
7.4.3 The phase of each wave determines
whether it reinforces or cancels the waves
contributed by the other spots.
7.4.4 Phases can be deduced from the wellunderstood diffraction patterns produced by
heavy-atom reference markers such as uranium
or mercury at specific sites in the protein.
7.4.5 Three-dimensional electron-density
distribution (map) is calculated by computers,
which gives the density of electrons at a large
number of regularly spaced points in the crystal.
7.4.6 Atomic structure of the protein is
obtained by interpreting the electron density map.
7.4.7 Resolution is determined by the
number of scattered intensities used in the
Fourier transformation. A higher number of
reflections (spots) corresponds to a higher
resolution.
7.4.8 Most protein structures are
determined between 3.0 to 2.0 Angstroms.
7.4.9 A (low) resolution of 6 Angstroms reveals the
course of the polypeptide chain, a (medium)
resolution of 2.8 Angstroms delineate groups of
atoms (chain tracing, identity of side chains and
some hydrogen bonds), a (medium-high)
resolution of 2.5 to 2.0 Angstroms shows phi, psi
angles, detailed side chain conformations,
hydrogen bonds, and bound water molecules), a
(very-high) resolution of 1.0 to 1.5 Angstroms
delineate individual atoms.
7.4.10 The ultimate resolution of an X-ray
analysis is determined by the degree of perfection
of the crystal of protein molecules, which is usually
at 2.0 Angstoms.
7.4.11 Atomic structures of hundreds of
proteins have been determined this way, providing
insight into how proteins recognize and bind other
molecules, how they function as enzymes, how they
fold and how they evolved.
7.5 Nuclear Magnetic Resonance (NMR)
spectroscopy can also reveal the atomic structure
of macromolecules in solution.
7.5.1 NMR can be used to determine
structures of proteins in solution.
7.5.2 The upper bound on size is about 30 kD,
because NMR peaks of larger proteins cannot be
adequately resolved and assigned at the current
time. Need higher magnetic field (now 800-1000
MHz).
7.5.3 NMR can be used to probe protein folding
and dynamics.
7.5.4 Sample preparation is also very difficult
to achieve, similar to X-ray crystallography. Need
very high solubility and concentration of proteins.