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Determining the sequence
One way: use an enzyme: Carboxypeptidase: hydrolyzes the peptide bond
(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 +
ser
asp
asp
ser
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METHODS . . .
AA mixture (ala, glu, lys
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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Handout 3-4
Side view
AAs applied at lower end
Isopropanol
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“front” =1.00
“Rf”
0.82
After stopping the paper
chromatography and staining
for the amino acids:
0.69
0.45
0.27
Most hydrophobic = furthest
Most hydrophilic = least far
0.11
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Paper chromatography apparatus
(felt tip black marker ink demonstration)
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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
Polypeptide chain
“Sub-peptides”
Determine sequence of each
subpeptide using the
carboxypeptidase technique
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The order of the subpeptides is unknown.
The sequence is reconstructed by noting the overlap between
differently produced subpeptides
Trypsin (lys, arg)
(1)
Chymotrypsin (trp, tyr, phe)
(2)
N
C
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)
Hemoglobin protein
trypsin
Sub-peptides
No further digestion to amino acids; left as sub-peptides
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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 electrophoreses
Fairly 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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Hb protein
In fingerprinting,
these spots contain
peptides, not
amino acids
trypsin
The mixture of
all sub-peptides
formed
Less negatively
charged,
more hydrophobic
Negatively
charged
Sequence just
this peptide:
------valine-----(sickle)
Positively
charged
Single AA substitution
---glutamate--(normal)
More
hydrophobic
negatively
charged
positively
charged
More
hydrophilic
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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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3-dimensional structure of proteins
One given purified polypeptide
• 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.
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?
“Renaturation” of a hard-boiled egg
Denature
by heat
Cool, renature?
X
ovalbumin
Too long
Cool, entangled
to sort out
Tangle, gel.
Probably due
to non-productive
hydrophobic
interactions
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urea
H
H
H
O
||
N-C-N
H
chaotropic agent
used at very high concentrations (e.g., 7 M)
gentler, gradual denaturation, renaturation
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“Renaturation” of ribonuclease after urea
+ urea,
denature
-urea, renature
“native” ribonuclease
active enzyme
compact
??
denatured ribonuclease
inactive enzyme
random coil
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Slow denaturation of
ribonuclease by urea
O
||
Urea = H2N-C—NH2
Ribonuclease in the
bag is denatured
Now dialyze out the urea
Macromolecules (protein here) cannot
permeate bag material
Small molecules (H20, urea) can permeate.
Ribonuclease
RENATURES
in the absence of any
other material
Urea will move from areas of high concentration to areas of low concentration.
Christian Anfinsen:
PRIMARY STRUCTURE DETERMINES TERTIARY STRUCTURE.
+ urea,
denatures
- urea, renatures
“The Anfinsen Experiment”
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Julio Fernandez lab, CU: a modern version of the Anfinson experiment
Force needed to pull
Pull
Length
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Denaturation/renaturation of domains ofa protein (titin) using the atomic force microscope.
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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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Protein folding
Primary structure itself results in some folding constraints:
See bottom of handout 3-3
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And these 4 atoms are in
one plane (N central)
These 4 redatoms
are ininone
so 6 atoms
oneplane
plane
(C of C=O central)
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There’s still plenty of flexibility
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This graphic intentionally left blank.
Secondary structure: the alpha helix
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Amino acids shown
simplified, without
side chains and H’s.
Almost every N-H and C=O
group can participate
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Alpha helix depictions
C = grays
N = blue
O = red
Poly alanine
Side chains = -CH3 (lighter
gray)
H’s not shown
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Linus Pauling and a model of the alpha helix.1963
Secondary structure:
H-bond
AA residue
beta pleated sheet
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Beta sheet (i.e., beta pleated sheet)
antiparallel
antiparallel
parallel
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Beta-sheets
Anti-parallel
Parallel
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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.
Neither
This is a popular “ribbon” model
of protein structure. Get familiar
with it. The ribbons are stretches
of single polypeptide chains. A
single ribbon is NOT a sheet.
A beta sheet
3 alpha helices
These “ribbon” depictions do not show the side chains, only the backbone
Tertiary structure
(overall 3-D)
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ionic
hydrophobic
H-bond
cys
Ion - dipole
interaction
covalent
Van der Waals
In loop regions and in regions of secondary structure
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Disulfide bond formation
Disulfide bond
(covalent, strong)
Sulfhydryl group
R-CH2-SH
cysteine
+
HS-CH2-R
cysteine
½ O2
R-CH2-S-S-CH2-R + HOH
cystine
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).
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 electrons
Actually it’s the loss and gain of the electrons that constitutes
oxidation and reduction, respectively.
No catalyst is usually needed here.
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Overall 3-D structure of a polypeptide is tertiary structure
Stays intact in the jacuzzi at 37 deg C
Usually does not require the strong covalent disulfide bond
to maintain its 3-D structure
[Tuber mode]l
Protein structures are depicted in a variety of ways
Backbone only
Ribbon
Small molecule
bound
Drawing attention
to a few side groups
Continuous lines, ribbons=
backbone (not sheets)
Space-filing,
with surface charge
blue = +
red =
-
Space-filling
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Most proteins are organized into
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4o,
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QUATERNARY STRUCTURE
Monomeric protein (no quaternary structure)
Dimeric protein (a homodimer)
The usual
weak
bonds
Dimeric protein (a heterodimer)
Also called:
multimeric proteins
A heterotetramer
A heteropolymeric protein (large one)
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Hemoglobin
$
One protein
$ 
Four polypeptide chains,
2 identical alphas
and 2 identical betas
Four “subunits”
Molecular weight

$

16,000
Subunit molecular weight
16,000
Subunit molecular weight
64,000
Protein molecular weight
$
$ 
64,000, even though the 4 chains are
not covalently bonded to each other
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Tetramer
Two heavy chains (H),
Two light chains (L)
Interchain disulfide bonds
The 4 weak bond types
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Sickle cell disease
Normal
glu
glu
Sickle cell
glu
glu
val
val val
val
Some small molecules can by added to a protein via covalent bonds.
One form of a “prosthetic group”.
Pyridoxal
phosphate
= Vitamin B6
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Most prosthetic groups are bound tightly via weak bonds.
Tetrahydrofolic acid
~ vitamin B9
Riboflavin
~ vitamin B2
Heme
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Membrane proteins
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Membrane 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
Small molecules bind with great specificity to pockets on protein surfaces
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Too far
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Estrogen receptor binding estrogen, a steroid hormone
detail
estrogen
estrogen
Estrogen receptor is specific, does not bind testosterone
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Protein binding can be very specific
Testosterone
Estrogen