Transcript Document

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}
Ester (functional group,
acid + alcohol)
Handout 2-9 top
A trigyceride (fat)
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Effect of fatty acid structure on physical properties
Saturated fatty acid
Solid fats
cis
Unsaturated fatty acid
cis
Oils
trans
trans
cis
X
No free rotation
No free
about rotation
double bonds
about double bonds
Hydrogenation of oil to solidify it
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H
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C
C
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X
H
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H
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C
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H
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C
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Free rotation
Free
rotation
about
single bonds
about single bonds
- 2H
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H
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C
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H
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H
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C
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H
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Adipose tissue
Adipocyte (fat storage cell)
Nuc.
Fat
globule
Fat is a good compact source of energy,
about twice the calories as starch, pound for pound.
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Phospholipids:
[HO]
[HO]
Handout 2-9
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O
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HO-P-O|
OPhosphoric acid
(phosphate ion)
+
R-OH
an alcohol
(hydroxyl)
O
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R-O-P-OH
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Oa phosphoester
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}
If R=another alcohol:
a phospho-diester
x
y
If R=H, phosphatydic acid
(2 FAs implied)
HO
HO –CH2CH2N+H3
HO
(alcohol = ethanolamine)
 phophatydyl ethanolamine
Handout 2-9
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HOH
2 fatty acid
tails each
Phosphate head
Biological membranes are phospholipid bilayers
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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
Phosphodiester
And:
Glycosidic bonds
C=C double bonds (cis and trans)
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PROTEINS
Amino acids (the monomer of proteins)
R = ONE of 20 CHEMICAL GROUPS
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At pH 7, ,most amino acids are zwitterions
(charged, but electrically neutral)
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pH7
+H+
Equilibrium state of the carboxyl group lies far towards
the ionized molecule at pH7
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50-50 charged-uncharged
at ~ pH9 (=the pK)
+OH- ( = -H+)
R
OH
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/
+H N - C – C=O
3
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H
pH:
Net charge:
1
+1
R
O|
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H2N - C – C=O
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H
R
O|
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+H N - C – C=O
3
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H
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-1
7
0
+H+
50-50 charged-uncharged
at ~ pH2.5 (=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
The 2 amino groups and the carboxyl are assumed to be charged (understood)
even if unwritten.
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guanido
+1
H+  H+
~10% charged at pH7
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Ball and stick physical model of an amino acid
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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 Sadava text
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A
B
D
C
Any
compound
Mannose
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Condensation of amino acids to form a polypeptide
(must be catalyzed)
dehydration again
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Parts of a polypeptide chain
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The backbone is monotonous
It is the side chains that provide the variety
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“Polypeptides” vs. “proteins”
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Polypeptide = amino acids connected in a linear chain (polymer)
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Protein = a polypeptide or several associated polypeptides (discussed later)
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Often used synonymously
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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 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
. . . . and an English name.
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: 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 + ser
…. arg-leu-leu-val-gly-ala-gly-phe-trp-lys-glu +
asp
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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
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Handout 3-4
Side view
AAs applied at lower end
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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
0.11
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Paper chromatography apparatus
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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
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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
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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
(Vernon 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
E.g.:
+
-
-
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
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Protein fingerprinting
Hb
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
------glutamate----(normal)
More
hydrophilic
Negatively
charged
Positively
charged
Negatively
charged
Positively
charged
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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.
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 scaffold)
or does it reside in the primary structure itself?
“Renaturation” of a hard-boiled egg
Denature
by heat
Cool, renature?
X
ovalbumin
Too long
to sort out
Tangle, gel.
Probably due
to non-productive
hydrophobic
interactions
Cool, entangled
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urea
H
H
H
O
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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 pure 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
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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.
Urea will move from areas of high concentration
to areas of low concentration
Ribonuclease
RENATURES
in the absence of any
other material
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Christian Anfinsen:
PRIMARY STRUCTURE DETERMINES TERTIARY STRUCTURE.
+ urea,
denatures
- urea, renatures
“The Anfinsen Experiment”