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Incidentally, note the functional
groups we have met so far:
Hydroxyl
Amine
Amide
Carboxyl
Carbonyl
Aldehyde
Ketone
Ester: Carboxylic acid ester
Phosphoester
And:
Glycosidic bonds
C=C double bonds (cis and trans)
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PROTEINS
Amino acids (the monomer of proteins)
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At pH 7, ,most amino acids are zwitterions
(charged but electrically neutral)
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+OH- ( -H+)
Net charge
+H+
50-50 charged-uncharged at ~ pH2.5 (=the pK)
50-50 charged-uncharged at ~ pH9 (=the pK)
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Numbering (lettering) amino acids
ε-amino group
ε
δ
γ
β
Alpha-amino
Alpha-carboxyl
(attached to the α-carbon)
Alpha-carbon
Amino acid examples
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Molecular weights 75 – 203
(MW)
Glycine (gly)
Side chain = H
Smallest (75)
One neg. charge
β-carboxyl:
-CH2-COOH
Tryptophan (trp)
5+6 membered
rings
Hydrophobic, largest
(203)
Lysine (lys)
One pos. charge
ε-amino
Alanine (ala)
One carbon
(methyl group)
-CH3
Arginine
(arg, guanidino group)
One pos. charge
-(NH-C (NH2)NH2)+,
Aspartic acid
(asp, aspartate)
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Shown uncharged (as on exams)
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Amino acids in 3 dimensions
• Asymmetric carbon (4 different
groups attached)
• Stereoisomers
• Rotate polarized light
• Optical isomers
• Non-superimposable
• Mirror images
• L and D forms
From Purves text
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Mannose
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(Without showing the R-groups)
The backbone is monotonous
It is the side chains that provide the variety
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“Polypeptides” vs. “proteins”
•
Polypeptide = amino acids connected in a linear chain (polymer)
•
Protein = a polypeptide or several associated polypeptides (discussed later)
•
Often used synonymously
•
Peptide (as opposed to polypeptide) is smaller, even 2 AAs (dipeptide)
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The backbone is monotonous
(Without showing the R-groups)
It is the side chains that provide the variety
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Proteins do most of the the jobs in the cell
E.g., egg albumin, hemoglobin, keratin, estrogen receptor,
immunoglobulins (antibodies), enzymes (e.g., beta-galactosidase)
Each is a polymer or assemblage of polymers made up of amino acids
Each particular protein polymer (polypeptide) has a unique sequence of amino acids
Each molecule of a particular protein has the same sequence of amino acids.
E.g., met-ala-leu-leu-arg-glu-leu-val- . . . .
How is this sequence determined?
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Primary (1o) Structure =
the sequence of the amino acids in the polypeptide chain
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Determining the sequence
One way:
enzyme:
Carboxypeptidase: hydrolyzes the peptide bond
,
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
asp
ser
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AA mixture
(-)
(+)
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A paper electrophoresis apparatus
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Side view
AAs applied at lower end
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“Rf”
0.82
After stopping the paper
chromatography and staining
for the amino acids:
0.69
0.45
0.27
0.11
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Paper chromatography apparatus
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• Treatment of a polypeptide with trypsin
• Trypsin is a proteolytic enzyme.
• It catalyzes cleavage (hydrolysis) after lysine and arginine residues
Polypeptide chain
Sub-peptides
The order of the subpeptides is unknown.
The sequence is reconstructed by noting the overlap between differently produced
subpeptides
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Trypsin (lys, arg)
(1)
Chymotrypsin (trp, tyr, phe)
(2)
N
C
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Fingerprinting a protein: analysis of the sub-peptides
(without breaking them down to their constituent amino acids)
Application to sickle cell disease
(Ingram, 1960’s)
Hemoglobin protein
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
+
H
H
H
-
Net charge = -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
Hb
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In fingerprinting,
these spots contain
peptides, not
amino acids
trypsin
The mixture of
all sub-peptides
formed
Less negatively charged,
More hydrophobic
Negatively
charged
---valine--(sickle)
Positively
charged
More
hydrophobic
More
hydrophilic
Negatively
charged
Positively
charged
Negatively
charged
Positively
charged
---glutamate--(normal)
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Every different polypeptide has a different primary structure (sequence).
Every polypeptide will have different arrangement of spots after fingerprinting.
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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
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3-D structure of polypeptides
Arrangement 1
at one moment
in time?
Arrangement 2
at one moment
in time?
Arrangement 3
at one moment
in time?
Arrangement 3
at one moment
in time?
[Rope models here]
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Got this far
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Primary structure itself results in some folding constraints:
See bottom of handout 3-3
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4 atoms in one plane
 6 atoms in one plane
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There’s still plenty of flexibility
Secondary structure: the alpha helix
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Amino acids shown
simplified, without
side chains and H’s.
Alpha helix depictions
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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
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Beta sheet
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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.
Tertiary structure: The overall 3-D structure of a polypeptide.
Neither
Beta-sheets
Alpha-helices
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
Ionic-H
combo covalent
Van der Waals
Weak bonds also occur throughout the polypeptide, between
the amino acid side chains in regions of secondary structure
as well as the looped regions
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Disulfide bond formation
Disulfide bond
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.
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 usually needed.
Overall 3-D structure of a polypeptide is tertiary structure
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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
Tuber model
Protein structures are depicted in a variety of ways
Backbone only
Space-filing,
With surface charge
Ribbon
Space-filling
Blue = negative charge
Red = positive
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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 in the primary structure itself?