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 (mol) 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 c

oncentration 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 =6r, 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 SDS-PAGE 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 SDS-PAGE 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-) /f3.2.2 Sedimentation coefficients are usually ex

pressed 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 (cross-reacting).

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 antibody-producing 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 enzyme-linked 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 proteins6.1 Proteins are optically active because they are dissymmetric (i.e., they cannot be superimposed onto their mirror images).

6.1.1 Asymmetric centers (the -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 -helices, structure motifs and random coils have been defined by using synthetic polypeptides and proteins of known structure.

6.3.3 The -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 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 rate-limiting 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 well-understood 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.