Determining the sequence

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Determining the sequence
Carboxypeptidase hydrolyzes the peptide bond
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-g
lu-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
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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
Isopropanol
AAs applied at lower end
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After stopping the paper chromatography
and staining for the amino acids
Rf 0.82 0.69 0.45 0.27 0.11
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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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
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)
N
C
(2)
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, 1960s)
trypsin
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Oligopeptides behave as a composite of their
constituent amino acids

-
-
Net charge -21 -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
Negatively charged
Positively charged
Single AA substitution
More hydrophobic
More hydrophilic
?negatively charged
positively? charged
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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?
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Renaturation of a hard-boiled egg
Denature by heat
X
Cool, renature?
ovalbumin
Too long to sort out
Cool, entangled
Tangle, gel. Probably due to non-productive hydro
phobic interactions
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urea

• 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
Ribonuclease in the bag is denatured
O Urea
H2N-CNH2
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.
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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
Relax force, re-contracts, renatures
Pull
Relax force, re-contracts, renatures
Force needed to pull
Length
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Denaturation/renaturation of domains ofa protein
(titin) using the atomic force microscope.
Julio Fernandez and colleagues, Columbia Univ.
Force applied
Length
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BUTChaperonins (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 membranes 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 in one plane (C of CO
central)
? so 6 atoms in one plane
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Theres still plenty of flexibility
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• This graphic intentionally left blank.

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Secondary structure the alpha helix
Amino acids shown simplified, without side
chains and Hs.
H
Almost every N-H and CO group can participate
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Alpha helix depictions
C grays N blue O red
Poly alanine Side chains -CH3 (lighter
gray) Hs not shown
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Linus Pauling and a model of the alpha helix.1963
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beta pleated sheet
Secondary structure
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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.
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.
These ribbon depictions do not show the side
chains, only the backbone
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Tertiary structure (overall 3-D)
ionic
hydrophobic
H-bond
cys
Ion - dipoleinteraction
covalent
Van der Waals
In loop regions and in regions of secondary
structure
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Disulfide bond formation
Sulfhydryl group
R-CH2-SH
HS-CH2-R
R-CH2-S-S-CH2-R
HOH

cystine
cysteine
cysteine
Two sulfhydryls have been oxidized (lost
Hs) Oxygen has been reduced (gained Hs). Oxygen
was the oxidizing agent (acceptor of the Hs).
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 its 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 model
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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-filling
Space-filing, with surface charge
blue red -
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Most proteins are organized into
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4o, QUATERNARY STRUCTURE
Monomeric protein (no quaternary structure)
Dimeric protein (a homodimer)
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
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Sickle cell disease
Normal
Sickle cell
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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
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Small molecules bind with great specificity to
pockets on protein surfaces
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
Estrogen
Testosterone