Download Lecture 9: Protein purification

Lecture 9: Protein purification
– Protein structure
– Use tables in book (Voet and Voet) for the
properties of peptidases for peptide sequencing
– Answer key for HW 2 posted
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Figure 6-25
Logarithmic relationship
between the molecular mass of a protein and its
relative electrophoretic mobility in SDS-PAGE.
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Figure 6-23
Detection of proteins by
immunoblotting.
Isoelectric focusing
• For looking at proteins without charge, proteins can be treated with 6M
urea (denatures but unlike SDS does not put charges on a protein).
• Thus, a mixture of proteins can be electrophoresed through a solution
having a a stable pH gradient in from the anode to the cathode and a
each protein will migrate to the position in the pH gradient according to
its isoelectric point. This is called isoelectric focusing.
• Ampholytes (amphoteric electrolytes)-low molecular mass (600900D) ooligomers with aliphatic amino and carboxylic acid groups with
a range of isoelectric points. Ampholytes help maintain the pH
gradiennt in the presence of high voltage.
• Can also use gels with immobilized pH gradients -made of
acrylamide derivatives that are covalently linked to ampholytes. Used
with a gradient maker to ensure continuously varied mixture when the
gel is made.
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Figure 6-26
General formula of the
ampholytes used in isoelectric focusing.
Isoelectric focusing
• 2D-gel electrophoresis is an invalubale tool for proteomics.
• Proteome (like genome) is the total number of all proteins expressed
by a cell or organism, but with an emphasis on their quantitation,
localization, modifications, interactions, and activities, as well as their
identification.
• Individual protein bands froma stained gel can be cut out of a gel,
destained, and and the protein can be eluted from the gel fragment for
identification and characterization using mass spec.
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Figure 6-27
Two-dimensional (2D) gel
electrophoresis.
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Table 6-1 Isoelectric Points of Several
Common Proteins.
Summary of techniques for protein
purification
•
•
•
Cell lysis techniques - osmolysis, mechanical disruption-high speed
blender, homogenizer, French press, sonication
Salting out and salting in
Chromatography
–
–
–
–
•
•
Ion exchange
Size exclusion
Affinity
others
Dialysis
Electrophoresis
– SDS PAGE
•
Isoelectric focusing
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Crystallization
Crystallization
•
•
•
•
Crystallization of proteinsdifficult.
Protein must be homogeneous
(e.g. pure)
Supersaturated solution prepared
(10 mg/ml) and allowed to stand
until crystals form.
Use x-ray diffraction to observe the
bonds that hold the 3-D shape of
the protein.
3-D structure of proteins
X-ray source
Single
crystal of
protein
Diffraction pattern
Computational
recombination of
scattered x-rays
Structural model
Electron density map
Figure 8-35
X-Ray diffraction photograph
of a single crystal of sperm whale myoglobin.
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Figure 8-36a Electron
density maps of proteins.
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Figure 8-36b Electron
density maps of proteins.
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Figure 8-36c Electron
density maps of proteins.
Molecular Recognition: The ability of molecules to
recognize and interact (bind) to specifically with other
molecules.
1. Forces of biological recognition are noncovalent,
relatively weak:
(1-30 kJ/mol) vs. 350 kJ/mol for carboncarbon single bond.
2. Noncovalent interactions are reversible.
==>Binding can start and stop within a short
timeframe.
3. Binding between molecules is specific.
• ==> Molecules only selectively bind other
molecules.
Noncovalent Bonding in Biomolecules
• Weaker interactions, termed non-covalent bonds,
often control folding, resulting shape and bring
biomolecules together for specific purposes.
• Ligand (L) + macromolecule (M-protein)
L + M  LM
Four types of weak, noncovalent bonds that can be involved
in molecular recognition between proteins, nucleic acids and
small molecules (e.g., substrates, drugs).
1. Van der Waals forces
2. Ionic bonds
(electrostatic forces)
3. Hydrogen bonds
4. Hydrophobic
interactions.
Figure 3.1 The Structure of the water molecule showing
the relative size of each atom by the Van der Waals radius.
• Covalent bonds hold
together oxygen and
hydrogen atoms.
• The polar character,
which is the result of
electronegativity
differences between
oxygen and hydrogen, is
indicated by the partial
charges (d+ and d-) on
atoms.
(b) Water has a dipole
moment because of its
bent geometry. The arrows
pointing to the more
electronegative atom are
used to show bond
polarity.
Figure 3.2 The CO2 molecule, although composed of
polar bonds, has no dipole moment because it is
linear.
The electronegativity differences between C and
O atoms are indicated by the partial charges (d).
Figure 3.3 Hydrogen bond between two water
molecules.
The hydrogen atom (partially charged) of one water
molecule interacts with a lone pair of electrons in an orbital
of the oxygen atom of another water molecule.
Figure 3.3
Hydrogen bonds
of biological
importance:
(a) between an alcohol and
water or between
alcohol molecules;
(b) between a carbonyl
group and water (X = H,
R, OH, OR, or NH2);
(c) between two peptide
chains, the carbonyl
group of one peptide
bonds to an N-H of
another;
(d) between complementary
base pairs in DNA.
Figure 3.5 The network of hydrogen bonds in water.
(a) The center water molecule may form hydrogen bonds with up to four
neighboring molecules, but the average is about three. The network structure is
constantly changing, with water molecules undergoing geometrical reorientations
and forming new hydrogen bonds with other neighboring water molecules.
Key point:
Water has a very high boiling point, freezing point, viscosity, surface
tension, and heat capacity relative to other simple non-metallic compounds
of similar molecular mass (weight).
Figure 3.6 Chemicals are made soluble in water by (a) dipoledipole interactions.
The partially charged positive atoms (hydrogen) of water and alcohol are
attracted to oxygen atom dipoles of water and alcohol. The carbonyl group
of an aldehyde, ketone, or acid can also be solvated by water.
Figure 3.6 Chemicals are made soluble in water by (a) dipoledipole interactions.
(b) Ion-dipole interactions. The positively charged sodium ion is surrounded
by molecules projecting their partially negative oxygen atoms (dipoles).
The acetate ion interacts with the partially positive hydrogen atoms (dipoles)
of water.
The highly polar nature of water allows it to break apart
(dissolve) ionic interactions that hold together many types
of salt crystals (NaCl)
Figure 3.5 The network of hydrogen bonds in water.
• A water molecule “wants” to form as many hydrogen bonds possible with its
neighbors to lower the net Gibbs free energy of the system.
• Typically 3-4 H-bonds possible in solution, transient cluster formation.
• The network structure is constantly changing, with water molecules undergoing
geometrical reorientations and forming new hydrogen bonds with other
neighboring water molecules.
Figure 3.7 The “Hydrophobic Effect”
•Because hydrophobic molecules have no polar groups to interact
with water, they have to be surrounded by a boundary of water
molecules.
•The formation of this highly ordered cage of water requires much
energy, which comes from hydrophobic interactions.
Water molecules form a highly ordered, low entropy Hbonded “cage” around nonpolar solute molecules
Hydrophobic Interactions
• A nonpolar solute "organizes" water.
• The H-bond network of water reorganizes to
accommodate the nonpolar solute (hydrocarbon).
• Fewer H-bonds are formed than desired, H2O molecules
must form a “cage” around nonpolar molecules.
• This is an increase in "order" of water.
• This is a decrease in ENTROPY (S).
• Gibbs Free Energy: DG = DH - TDS
• A decrease in entropy (-S) results in an increase in
Gibbs free energy (+G)!
Figure 3.8 Formation of a
micelle from the sodium
salt of a long chain
carboxylic acid.
The nonpolar
hydrocarbon tails of the
acid arrange themselves
to avoid contact with
water.
The negatively charged
carboxyl groups interact
with water by forming
ion-dipole interactions.
Structural hierarchy in proteins
• Primary structure (1º structure)-for a protein is the
amino acid sequence of its polypeptide chain(s).
• Secondary structure (2º structure)-the local spatial
arrangement of a polypeptide’s backbone atoms without
regard to the conformations of their side chains.
• Tertiary structure (3º structure)-refers to the 3dimensional structure of an entire polypeptide (close to
secondary structure).
• Quaternary structure (4º structure)-The spatial
arrangement of a protein’s subunits
– Most protein is made up of two or more polypeptide chains
(subunits) associated through noncovalent interactions.
Structural hierarchy in proteins
Amide character in the peptide bond
+
H3N
R1 O
R2 O
C
N
C
H
H
H
C
C
O-
• Peptides are rigid due to resonance around the amide bond, having
≈ 40% double-bond character.
• This restricts the rotation due to delocalization of electrons and
overlap of the O-C-N  orbitals.
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Figure 8-1The trans-peptide group.
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Figure 8-2 The cis-peptide
group.
Amide character in the peptide bond
• The double bond character results in a planar form around the
peptide bond.
• Because the backbone of the
peptide bond is planar, the
backbone conformation can be
specified by the torsion angles
(rotation angles, dihedral angels)
about the C-N bond () and the CC bond () of each amino acid.
  and  are both defined as 180º
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when the polypeptide chain is
planar, fully extended (all trans)
conformation and increase for a
clockwise rotation when viewed from
C 
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Figure 8-5 Conformations of
ethane.
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Figure 8-6 Steric interference
between adjacent residues.
Ramachandran Diagrams
• Show the allowed conformations of polypeptides.
• These work, because the sterically allowed values of 
and  can be determined by calculating the distances
between the atoms at all values  and  for the central
peptide unit.
• Sterically forbidden conformations are those in which
any nonbonding interatomic distance is less than its
corresponding van der Waals distance.
• This info can be summarized by the Ramachandran
Diagram or Conformation map
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Structural properties predicted by
Ramachandran Diagram
Regions of “normally allowed” torsion
angles are shown in blue.
Green regions are the “outer limit”
regions.
Secondary structure
 (deg)
 (deg)
Right handed  helix ()
-57
-47
Parallel  pleated sheet ()
-119
113
Antiparallel  pleated sheet ()
-139
135
Right-handed 310 helix (3)
-49
-26
Right-handed helix ()
-57
-70
2.27 ribbon (2)
-78
59
Left-handed polyglycine II and
poly-L-proline II helices (II)
-79
150
Collagen (C)
-51
151
Left-handed  helix (L)
57
47
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Table 8-1 van der Waals Distances for
Interatomic Contacts.
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Figure 8-8 Conformation
angles in proteins.
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+
H3N
COOC
R
H
+
H3N
COO-
C
H
H