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Determining the sequence
One way: use an enzyme: (an old method, but useful for teaching)
identify,
e.g., …. arg-leu-leu-val-gly-ala-gly-phe-trp-lys-glu-asp-ser
…. arg-leu-leu-val-gly-ala-gly-phe-trp-lys-glu-asp +
…. arg-leu-leu-val-gly-ala-gly-phe-trp-lys-glu +
Carboxypeptidase: hydrolyzes the peptide bond
ser
asp
ser asp
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(-) (+)
AA mixture (ala, glu, lys
METHODS . . .
Anode Cathode
Note: The cathode is negative in an electrophoresis apparatus even though it is positive in a battery (voltaic cell).
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A paper electrophoresis apparatus
2000 to 4000 volts DC, dangerous
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AAs applied at lower end
Side view
Handout 3-4
Isopropanol
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“Rf”
0.82
0.69
0.45
0.27
0.11
After stopping the paper chromatography and staining for the amino acids:
1.00“front” =
Most hydrophobic = furthest
Most hydrophilic = least far
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Paper chromatography apparatus
(felt tip black marker ink demonstration)
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“Sub-peptides”
Polypeptide chain
Ordering the sub-peptides within the polypeptifde:
• Treatment of a polypeptide with trypsin• Trypsin is a proteolytic enzyme.• It catalyzes cleavage (hydrolysis) after lysine and arginine residues
Determine sequence of eachsubpeptide using the carboxypeptidase technique
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N C
Trypsin (lys, arg)
Chymotrypsin (trp, tyr, phe)
The order of the subpeptides is unknown.The sequence is reconstructed by noting the overlap between differently produced subpeptides
(1)
(2)
Sequence overlap
Done!
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Quick way to compare two proteins without sequencing:Fingerprinting a protein: analysis of the sub-peptides themselves.(Without sequencing, i.e., without breaking them down to their constituent amino acids)
Application to sickle cell disease(Vernon Ingram, 1960’s)
Sub-peptides
Hemoglobin protein
No further digestion to amino acids; left as sub-peptides
trypsin
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Oligopeptides behave as a composite of their constituent amino acids
Net charge = -2+1= -1: moves toward the anode in paper electrophoresesFairly hydrophobic (~5/6): expected to move moderately well in paper chromatography
Nomenclature: ala-tyr-glu-pro-val-trp or AYEPVW
or alanyl-tyrosyl-glutamyl-prolyl-valyl-tryptophan
+
-
-
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In fingerprinting, these spots containpeptides, not amino acids
The mixture of all sub-peptides formed
More hydrophobic
More hydrophilic
Negativelycharged
Positivelycharged
negatively charged
positivelycharged
Less negatively charged,more hydrophobic
Hb protein
trypsin
---glutamate--- (normal)
------valine------(sickle)
Sequence just this peptide:
Single AA substitution
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Every different polypeptide has a different primary structure (sequence). By definiton.
The migration behavior of each sub-peptide depends on its composite properties.
The properties are suffiently complex such that most subpeptides in a given polypeptide will behave differently.
Every polypeptide will have different arrangement of spots after fingerprinting.
Four polypeptide fingerprints
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• Molecule #1: N-met-leu-ala-asp-val-val-lys-....
• Molecule #2: N-met-leu-ala-asp-val-val-lys-...
• Molecule #3: N-met-leu-ala-asp-val-val-lys-...
• Molecule #4: N-met-leu-ala-asp-val-val-lys-... etc.
3-dimensional structure of proteins
One given purified polypeptide
clothesline . . .
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Information for proper exact folding(How does a polypeptide fold correctly?)
Predicting protein 3-dimensional structure
Determining protein 3-dimensional structure
Where is the information for choosing the correct folded structure?
Is it being provided by another source (e.g., a template)or does it reside in the primary structure itself?
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Denature by heat
Cool, renature?
Tangle, gel.Probably due to non-productivehydrophobic interactions
XToo long to sort out
“Renaturation” of a hard-boiled egg
ovalbuminCool, entangled
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urea
chaotropic agent
used at very high concentrations (e.g., 7 M)
gentler, gradual denaturation, renaturation
O||
N-C-N
H
H
H
H
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+ urea, denature
-urea, renature
??
“Renaturation” of ribonuclease after urea
“native” ribonucleaseactive enzyme
compact
denatured ribonucleaseinactive enzyme
random coil
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Now dialyze out the urea
Slow denaturation of ribonuclease by urea
O ||Urea = H2N-C—NH2
Macromolecules (protein here) cannot permeate bag material
Small molecules (H20, urea) can permeate.
Urea will move from areas of high concentration to areas of low concentration.
Ribonuclease in the bag is denatured
RENATURESRibonuclease
in the absence of any other material
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PRIMARY STRUCTURE DETERMINES TERTIARY STRUCTURE.
Christian Anfinsen:
+ urea, denatures
- urea, renatures
“The Anfinsen Experiment”
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20Julio Fernandez lab, CU: a modern version of the Anfinson experiment
For
ce n
eede
d to
pul
lRelax force, re-contracts, renatures Pull
Length
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Denaturation/renaturation of domains ofa protein (titin) using the atomic force microscope.
Julio Fernandez and colleagues, Columbia Univ.
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BUT:Chaperonins
(made of proteins themselves)
• Help fold proteins during synthesis
• Perhaps by preventing illegitimate interactions, like intermolecular contacts via exposed hydrophobic groups of partially folded proteins
• Also help re-fold proteins that have denatured after passing through a membrane’s P-lipid bilayer, e.g., during transport into a mitochondrion (organelle).
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Zsolt Török, Laszlo Vigh and Pierre Goloubinoff, 1996 The Journal of Biological Chemistry, 271, 16180-16186.
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See bottom of handout 3-3
Primary structure itself results in some folding constraints:
Protein folding
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These 4 red atoms are in one plane(C of C=O central)
so 6 atoms in one plane
And these 4 atoms are in one plane (N central)
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There’s still plenty of flexibility
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This graphic intentionally left blank.
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Amino acids shown simplified, without side chains and H’s.
Secondary structure: the alpha helix
Almost every N-H and C=O group can participate
H
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Poly alanineSide chains = -CH3 (lighter gray)H’s not shown
C = graysN = blueO = red
Alpha helix depictions
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Linus Pauling and a model of the alpha helix.1963
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AA residue
H-bond
Secondary structure: beta pleated sheet
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Beta sheet (i.e., beta pleated sheet)
antiparallel
parallel
antiparallel
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Anti-parallel Parallel
Beta-sheets
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secondary structure (my definition):
structure produced by regular repeated interactions between atoms of the backbone.
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Tertiary structure: The overall 3-D structure of a polypeptide.
These “ribbon” depictions do not show the side chains, only the backbone
3 alpha helices
This is a popular “ribbon” modelof protein structure. Get familiar with it. The ribbons are stretchesof single polypeptide chains. A single ribbon is NOT a sheet.
A beta sheet
Neither
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41Tertiary structure
(overall 3-D)
ionic
hydrophobic
H-bond
Ion - dipoleinteraction
Van der Waals
cys
covalent
In loop regions and in regions of secondary structure
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R-CH2-SH HS-CH2-R+ R-CH2-S-S-CH2-R½ O2
+ HOH
cysteine cysteine cystine
Disulfide bond formation
An oxidation-reduction reaction: Cysteines are getting oxidized (losing H atoms, with electron; NOT losing a proton, not like acids.)Oxygen is getting reduced, gaining H-atoms and electronsActually it’s the loss and gain of the electrons that constitutes oxidation and reduction, respectively. No catalyst is usually needed here.
Sulfhydryl groupDisulfide bond
(covalent, strong)
Two sulfhydryls have been oxidized (lost H’s)Oxygen has been reduced (gained H’s).Oxygen was the oxidizing agent (acceptor of the H’s).
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Stays intact in the jacuzzi at 37 deg C
Usually does not require the strong covalent disulfide bond to maintain its 3-D structure
Overall 3-D structure of a polypeptide is tertiary structure
[Tuber mode]l
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Space-filing,with surface charge
Backbone only
Protein structures are depicted in a variety of ways
Ribbon
Space-filling
Small molecule bound
blue = +
red = -
Continuous lines, ribbons=backbone (not sheets)
Drawing attention to a few side groups
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Most proteins are organized into
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474o, QUATERNARY STRUCTURE
Monomeric protein (no quaternary structure)
Dimeric protein (a homodimer)
Dimeric protein (a heterodimer)
A heterotetramer
A heteropolymeric protein (large one)
Also called: multimeric proteins
The usual weak bonds
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Hemoglobin
Molecular weight
16,000
16,000
64,000
Subunit molecular weight
Subunit molecular weight
Protein molecular weight
One protein
Four polypeptide chains, 2 identical alphas and 2 identical betasFour “subunits”
64,000, even though the 4 chains are not covalently bonded to each other
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Two heavy chains (H),Two light chains (L)
Interchain disulfide bonds
Tetramer
The 4 weak bond types
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Normal Sickle cell
glu glugluglu valval valval
Sickle cell disease
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Pyridoxal phosphate
Some small molecules can by added to a protein via covalent bonds.One form of a “prosthetic group”.
= Vitamin B6
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Riboflavin~ vitamin B2
Heme
Tetrahydrofolic acid~ vitamin B9
Most prosthetic groups are bound tightly via weak bonds.
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Membrane proteins
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54Membrane proteins
Hydrophobic side chains on the protein exterior for the portion in contact with the interior of the phospholipid bilayer.
Anions are negatively charged.
Cations are positively charged
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Too far
Small molecules bind with great specificity to pockets on protein surfaces
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57Estrogen receptor binding estrogen, a steroid hormone
estrogen estrogen
detail
Estrogen receptor is specific, does not bind testosterone
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58Protein binding can be very specific
Testosterone Estrogen