Post on 05-Apr-2018
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Amino Acids
& Proteins
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Amino Acids
Amino acid: A compound that contains both an
amino group and a carboxyl group. -Amino acid: An amino acid in which the amino group
is on the carbon adjacent to the carboxyl group.
although -amino acids are commonly written in the
unionized form, they are more properly written in thezwitterion (internal salt) form.
RCHCOH
NH2
RCHCO-
NH3+
-Amino Acid Zwitterion
form
OO
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Chirality of Amino Acids
With the exception of glycine, all protein-derived
amino acids have at least one stereocenter (the-carbon) and are chiral.
the vast majority have the L-configuration at their -carbon.
COO-
CH3
HH3N
L-Alanine
COO-
CH3
H NH3+
D-Alanine
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Amino Acids with Nonpolar side chains
NH3+
COO
-
NH3+
COO-
NH3+
COO-
NH3+
COO-
NH3+
COO-S
NH3+
COO-
N
H H
COO-
NH3+
COO-
NH
COO-
NH3+
Alanine(Ala, A)
Glycine
(Gly, G)
Isoleucine
(Ile, I)
Leucine
(Leu, L)
Methionine
(Met, M)
Phenylalanine(Phe, F)
Proline
(Pro, P)
Tryptophan
(Trp, W)
Valine(Val, V)
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With Polar side chains
NH3+
COO-H2N
O
NH3+
COO-H
2
N
O
NH3+
COO-HO
NH3 +
COO-OH
Asparagine
(Asn, N)
Glutamine
(Gln, Q)
Serine
(Ser, S)
Threonine
(Thr, T)
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With Acidic & Basic Side Chains
NH3+
COO--O
O
NH3+
COO-
-O
O
NH3+
COO-HS
NH3+
COO-
HO
NH3+
COO-NH
H2N
NH2+
NH3+
COO-N
NH
NH3+
COO-H3N
Cysteine
(Cys, C)
Tyrosine
(Tyr, Y)
Glutamic acid(Glu, E)
Aspartic acid(Asp, D)
Histidine
(His, H)
Lysine
(Lys, K)
Arginine
(Arg, R)
+
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Acid-Base properties, Non polar and polar side chains
pKa
ofpKa
of
valine 2.29 9.72
tryptophan 2.38 9.39
9.102.09threonine
serine 2.21 9.1510.602.00proline
phenylalanine 2.58 9.24
9.212.28methionine
9.742.33leucine
isoleucine 2.32 9.76glycine 2.35 9.78
9.132.17glutamine
8.802.02asparagine
9.872.35alanine
Nonpolar &
polar sidechains NH3
+COOH
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Acid-Base Properties, Acidic/Basic Side Chains
pKa ofpKa ofpKa of
10.079.112.20tyrosine
lys ine 2.18 8.95 10.53
6.109.181.77histidine
glutamic acid 2.10 9.47 4.07
8.0010.252.05cysteine
aspartic acid 2.10 9.82 3.86
arginine 2.01 9.04 12.48
SideChain
AcidicSideChains NH3
+COOH
pKa ofpKa ofpKa of Side
Chain
BasicSide
ChainsNH
3
+COOH
carboxyl
carboxyl
sufhydryl
phenolic
guanidino
imidazole
1 amino
SideChain
Group
SideChain
Group
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Acidity: -COOH Groups
The average pKa of an -carboxyl group is 2.19,
which makes them considerably stronger acidsthan acetic acid (pKa 4.76).
The greater acidity is accounted for by the electron-withdrawing inductive effect of the adjacent -NH3
+
group.
NH3+
RCHCOOH H2 O
NH3+
RCHCOO-
H3 O+
+ pKa = 2.19+
Note that the NH2 will be protonated at theselow pHs
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Acidity: side chain -COOH
Due to the electron-withdrawing inductive effect
of the -NH3+ group, side chain -COOH groupsare also stronger than acetic acid.
The effect decreases with distance from the -NH3+
group. Compare:
-COOH group of alanine (pKa 2.35)
-COOH group of aspartic acid (pKa 3.86)
-COOH group of glutamic acid (pKa 4.07)
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Acidity: -NH3+ groups
The average value of pKa for an -NH3+ group is
9.47, compared with a value of 10.76 for a 1alkylammonium ion.
NH3+
RCHCOO-
H2 O
NH3+
CH3CHCH3 H2 O
NH2
RCHCOO-
NH2
CH3CHCH3
H3 O+
H3 O+ pKa = 10.60++
+ pKa = 9.47+
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Details: The Guanidine Group of Arg
The basicity of the guanidine group is attributed to the
large resonance stabilization of the protonated formrelative to the neutral form.
CRNH
NH2+
NH2
C
NH2+
NH2
RNH
CRN
NH2
NH2
NH2
CRNH
NH2
H3 O+
H2 O
+
:
: :
:
:
:
:
:
+
pKa = 12.48
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Details: Imidazole Group
The imidazole group is a heterocyclic aromatic amine.
N
N
H
H
COO-
NH3+ N
N
H
H
COO-
NH3+
H2O
H3O+
H
COO-
NH3+
N
NH3O
+
Not a part of thearomatic sextet;
the proton acceptor pKa 6.10+
+
+
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Isoelectric Point
Isoelectric point (pI): pH at which an amino acid,
polypeptide, or protein has a total charge of zero. The pI for glycine, for example, falls between the pKa
values for the carboxyl and amino groups.
pI =12
( pKa COOH + pKa NH3+
)
=21 (2.35 + 9.78) = 6.06
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Isoelectric Point of glycine continued
pKa = pH-log([conj base]/[acid])
At pH = 6.06
For carboxyl group 2.35 = 6.06 - log ([RCO2-]/[RCO2H])
6.06- 2.35 = 3.71 = log ([RCO2-
]/[RCO2H])([RCO2
-]/[RCO2H]) = 103.71 or 99.98% ionized as neg ion.
For amino group 9.78 = 6.06 - log([RNH2]/[RNH3+])
([RNH2]/[RNH3+]) = 10-3.72 or 99.98% protonated.
On average Zero Chg.
pI =12
( pKa COOH + pKa NH3+
)
=21 (2.35 + 9.78) = 6.06
Again
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Furthermore..
[A+] = [C-]
A+ BC-
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Titration of conjugate acid of glycine with NaOH.
Buffer solution.[-CO2H] = [-CO2
-]
Strongly acid
-CO2H and -NH3+
Isoelectric.Zero netcharge.
Buffer solution.
[-NH3+]=[-NH2]
Strongly basic-CO2
- and NH2
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Isoelectric Point
6.11
5.41
5.65
6.06
6.04
6.04
5.74
5.91
6.30
5.685.60
5.88
6.00
pKa ofpKa
ofpKa
of
pI
----
----
--------
--------
----
----
----
----
----
--------
valine 2.29 9.72
tryptophan 2.38 9.39
9.102.09threonineserine 2.21 9.15
10.602.00proline
phenylalanine 2.58 9.24
9.212.28methionine
9.742.33leucine
isoleucine 2.32 9.76
glycine 2.35 9.78
9.132.17glutamine
8.802.02asparagine
9.872.35alanine
SideChain
Nonpolar &
polar sidechains NH3
+COOH
If pH is lower than pI then more protonated
molecules. If higher then more negative charge.
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Isoelectric Point
10.76
2.98
5.02
3.08
7.649.74
5.63
pKa ofpKa
ofpKa
of
pI
10.079.112.20tyrosine
lysine 2.18 8.95 10.53
6.109.181.77histidine
glutamic acid 2.10 9.47 4.07
8.0010.252.05cysteine
aspartic acid 2.10 9.82 3.86
arginine 2.01 9.04 12.48
SideChain
AcidicSide Chains NH3
+COOH
pKa ofpKa ofpKa of
pISideChain
BasicSide Chains NH3
+COOH
Three buffered pHs
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Aspartic acid
pKa 2.10 3.86 9.82
A+ B C- D2-
pI = (2.10 + 3.86)/2[A+] = [C-][D2-] approx 0
[A+] = [B]
pH = pKa
[B] = [C-]
Note species Bhas zero netcharge. pKa1 and
pKa2 control [A+]and [C-] whichshould be equal.
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Arginine
B+A2+ C D-
pI = (9.04+12.48)/2 =10.76[B+] = [D-]; [A2+] about 0
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Electrophoresis
Electrophoresis: The process of separating
compounds on the basis of their electric charge. electrophoresis of amino acids can be carried out
using paper, starch, polyacrylamide and agarose gels,and cellulose acetate as solid supports.
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Electrophoresis
A sample of amino acids is applied as a spot on the
paper strip. An electric potential is applied to the electrode vessels
and amino acids migrate toward the electrode withcharge opposite their own.
Molecules with a high charge density move faster thanthose with low charge density.
Molecules at isoelectric point remain at the origin.
After separation is complete, the strip is dried and
developed to make the separated amino acids visible. After derivitization with ninhydrin, 19 of the 20 amino
acids give the same purple-colored anion; prolinegives an orange-colored compound.
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Electrophoresis
The reagent commonly used to detect amino acid is
ninhydrin.
RCHCO-
O OH
O
O
OHNH3
+
O
O-
N
O
O
O
RCH CO2 H3 O++
An -aminoacid
Purple-colored anion
+ +
2+
Ninhydrin
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Polypeptides & Proteins
In 1902, Emil Fischer proposed that proteins are
long chains of amino acids joined by amidebonds to which he gave the name peptide bonds.
Peptide bond:The special name given to theamide bond between the -carboxyl group of oneamino acid and the -amino group of another.
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Serinylalanine (Ser-Ala)
H2N HO
O
HHOCH
2
Serine
(Ser, S)
H2N
O
OH
H CH3
Alanine
(Ala, A)
+
H2NN
OH
HOCH2
H
H
CH3O
H O
Serinylalanine
(Ser-Ala, (S-A)
peptide bond
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Peptides
Peptide:The name given to a short polymer of amino
acids joined by peptide bonds; they are classified bythe number of amino acids in the chain.
Dipeptide: A molecule containing two amino acidsjoined by a peptide bond.
Tripeptide: A molecule containing three amino acidsjoined by peptide bonds.
Polypeptide: A macromolecule containing many aminoacids joined by peptide bonds.
Protein: A biological macromolecule of molecularweight 5000 g/mol or greater, consisting of one ormore polypeptide chains.
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Writing Peptides
By convention, peptides are written from the left,
beginning with the free -NH3+
group and ending withthe free -COO- group on the right.
H3N
OH
NH
O
HN
COO-
O-
OC6 H5O
+
C-terminal
amino acid
N-terminal
amino acid
Ser-Phe-Asn
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Writing Peptides
The tetrapeptide Cys-Arg-Met-As
At pH 6.0, its net charge is +1.
HN N
HO
HN O
-
OO
O
NH2
SCH3
NH
NH2+
H2N
O
H3 N
SH C-terminalamino acid
N-terminal
amino acid
pKa 12.48
pKa
8.00
+
At pH 8 it would behalf ionized.
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Primary Structure
Primary structure: The sequence of amino acids
in a polypeptide chain; read from the N-terminalamino acid to the C-terminal amino acid:
Amino acid analysis:
Hydrolysis of the polypeptide, most commonly carriedout using 6M HCl at elevated temperature.
Quantitative analysis of the hydrolysate by ion-exchange chromatography.
C
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Ion Exchange Chromatography
Analysis of a
mixture of aminoacids by ionexchangechromatography
C B id B CN
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Cyanogen Bromide, BrCN
BrCN Selectively cleaves of peptide bonds formed by
the carboxyl group of methionine.
PN -C-NH CH C NH-PC
O O
CH2CH2 -S-CH3
cyanogen bromide isspecif ic for the cleavage
of this peptide bond
from theN-terminal
end
from theC-terminal end
C B id B CN
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Cyanogen Bromide, BrCN
O
S-CH3
HNC-NH
Opept ide COO-
H3 N pept ideBr C N
H2 O
O
OC-NH
O
H3 N pept ide
H3N pept ide COO-
CH3 S- C N
side chainof methionine
+0.1 M HCl
A substituted -lactone ofthe amino acid homoserine
Methylthiocyanate
Mechanism to follow
C B id B CN
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Cyanogen Bromide, BrCN
Step 1: Nucleophilic displacement of bromine.
O
S-CH3
H3 N C-NH
O HN COO-
Br C N
O
S-CH3C N
H3 N C-NH
O HN COO-
Br
Cyanogen bromide
a sulfoniumion; a good
leaving group
::
:
C B id B CN
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Cyanogen Bromide, BrCN
Step 2: Internal nucleophilic displacement of methyl
thiocyanate.
O
S-CH3
C N
HN COO-H3N C-NH
O
O
H3N C-NH
OHN COO-
CH3-S-C N
An iminolactonehydrobromide
+
Methylthiocyanate
:
:
:
:
C B id B CN
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Cyanogen Bromide, BrCN
Step 3: Hydrolysis of the imino group.
O
H3 N C-NH
OHN COO-
H2 O
OH3 N C-NH
O
O H3N COO-
A substituted -lactone of
the amino acid homoserine
E C t l i
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Enzyme Catalysis
A group of protein-cleaving enzymes called
proteases can be used to catalyze the hydrolysisof specific peptide bonds.
Phen ylalanine, tyrosin e, tryptophanChymotrypsin
Arginine, lysineTrypsin
Catalyzes Hydrolysis of Peptide BondFormed b y Carboxyl Group ofEnzyme
Ed D d ti
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Edman Degradation
Edman degradation: Cleaves the N-terminal
amino acid of a polypeptide chain.
S=C=N-Ph
H2 N COO-HN
N
O
R
SPh
H3 NNH
R
O
COO- +
+
N-terminal
amino acid
A phenylthiohydantoin
Phenyl isothiocyanate
+
Ed D d ti
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Edman Degradation
Step 1: Nucleophilic addition to the C=N group of
phenylisothiocyanate and proton tautomerization
H2 NNH
R
O
COO-
Ph N C S
HN
S
RO
NH COO-
Ph NH
A derivative of
N-phenylthiourea
:
::
Ed D d ti
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Edman Degradation
Step 2: Nucleophilic addition of sulfur to the C=O of
the adjacent amide group and acid catalysis.
HN
S
RO
NH COOHPh N
H
H
HN
S
R
O
Ph-N
HN
S
R
Ph-N
OH
NH
H
COOH
H+
H3N COOH
H+
+
A thiazolinone
+
+
+
Edman Degradation
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Edman Degradation
Step 3: Isomerization of the thiazolinone ring.
HN
S
R
O
Ph-N
+ H-Nu
R
HN
N SHNu
O
Ph
R
HN
S NH
Ph
Nu
O - H-NuHN
N
R
O
S
Ph
A thiazolinone
A phenylthiohydantoin
keto-enoltautomerism
substitution
DNFB tagging of N terminal AA
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DNFB tagging of N terminal AA
C b tid l f C t i l AA
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Carboxypeptidase cleavage of C terminal AA
Treatment of peptide with carboxypeptidase
cleaves the peptide linkage adjacent to the freealpha carboxyl group. It may then be identified.
Primary Structure example
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Primary Structure, example
Deduce the 1 structure of this pentapeptide
pentape ptide
Edman Degradation
Hydrolysis - Chymotryps in
Fragment A
Fragme nt B
Hydrolysis - Trypsin
Fragment C
Fragment D
Arg, Glu, His , Phe, Se r
Glu
Glu, His, Phe
Arg, Ser
Arg, Glu, His, Phe
Ser
Expe riment al Procedure Amino Acid Composition
Phen ylalanine, tyrosin e, tryptophanChymotrypsin
Arginine, lysineTrypsin
Catalyzes Hydrolysis of Peptide BondFormed b y Carboxyl Group ofEnzyme
using
Glu-
Glu-His-Phe
Glu-His-Phe-Arg-Ser
Polypeptide Synthesis
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Polypeptide Synthesis
The problem in protein synthesis is how to join
the -carboxyl group of aa-1 by an amide bond tothe -amino group of aa-2, and not vice versa.
aa1
H3NCHCO-
O
aa2
H3NCHCO-
O
aa1 aa2
H3NCHCNHCHCO-
OO
H2O?
+++
++
Polypeptide Synthesis Strategy
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Polypeptide Synthesis Strategy
Protect the -amino group of aa-1.
Activate the -carboxyl group of aa-1. Protect the -carboxyl group of aa-2.
+
+
form pe ptidebond
protectinggroup
activatinggroup
protectinggroup
O O
aa2aa1
Z-NHCHC- Y H2 NCHC-X
Z-NHCHCNHCHC-X H-Y
O O
aa1 aa2
Amino Protection
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Amino-Protection
convert them to amides.
O
( CH3
)3
COCOCOC(CH3
)3
O O
(CH3 )3COC-
O
PhCH2OC-
O
PhCH2OCCl
Di-t ert-butyl dicarbonate
Benzyloxycarbonylchloride
t ert -Butoxycarbonyl(BOC-) group
Benzyloxycarbonyl(Z-) group
Amino Protection
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Amino-Protection
Treatment of an amino group with either of these
reagents gives a carbamate (an ester of themonoamide of carbonic acid).
A carbamate is stable to dilute base but can beremoved by treatment with HBr in acetic acid.
PhCH2OCNH-peptideO HBr
CH3COOHPhCH2 Br CO2 H3 N-peptide
A Z-protectedpeptide
+ + +
Benzyl
bromide
Unprotected
peptide
PhCH2OCCl
O
CH3
H3 NCHCO-
O1. NaOH
2. HCl, H2 OCH3
PhCH2 OCNHCHCOH
O O
Alanine N-Benzyloxycarbonylalanine(Z-Ala)
++
Benzyloxycarbonylchloride (Z-Cl)
Amino Protecting Groups
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Amino-Protecting Groups
The benzyloxycarbonyl group is also removed by
hydrogenolysis. The intermediate carbamic acid loses carbon dioxide
to give the unprotected amino group.
PhCH2OCNH-peptide
O
H2Pd
PhCH3 CO2 H2 N-pept ide
A Z-protectedpeptide
Unprotected
peptide
Toluene+++
Carboxyl Protecting Groups Esters
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Carboxyl-Protecting Groups. Esters
Carboxyl groups are most often protected as
methyl, ethyl, or benzyl esters. Methyl and ethyl esters are prepared by Fischer
esterification, and removed by hydrolysis in aqueousbase under mild conditions.
Benzyl esters are removed by hydrogenolysis; theyare also removed by treatment with HBr in acetic acid
Peptide Bond Formation
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Peptide Bond Formation
The reagent most commonly used to bring about
peptide bond formation is DCC. DCC is the anhydride of a disubstituted urea and,
when treated with water, is converted to DCU.
1,3-Dicyclohexylcarbodiimide(DCC)
+
N,N' -dicyclohexylurea
(DCU)
C NN
H
N NC
H
O
H2 O
In bringing aboutformation of a peptidebond, DCC acts adehydrating agent.
Peptide Bond Formation
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Peptide Bond Formation
Carboxyl-prote cted
aa2
Amino-protected
aa1
++CHCl3
Z-NHCHC- OH H2 NCHCOCH3
Amino and carboxyl
protected dipeptide
+Z-NHCHC-NHCHCOCH3
DCC
DCU
R1 R2
R1 R2
O O
O O
Solid-Phase Synthesis
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Solid-Phase Synthesis
Bruce Merrifield, 1984 Nobel Prize for Chemistry
Solid support: a type of polystyrene in which about 5%of the phenyl groups carry a -CH2Cl group.
The amino-protected C-terminal amino acid is bondedas a benzyl ester to the support beads.
The polypeptide chain is then extended one aminoacid at a time from the N-terminal end.
When synthesis is completed, the polypeptide isreleased from the support beads by cleavage of the
benzyl ester.
Solid-Phase Synthesis
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Solid-Phase Synthesis
The solid support used in the Merrifield solid
phase synthesis.
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Solid-Phase Synthesis
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Solid-Phase Synthesis
Merrifield synthesized the
enzyme ribonuclease, aprotein containing 124 aminoacids
Peptide Bond Geometry
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Peptide Bond Geometry
The four atoms of a peptide bond and the two alpha
carbons joined to it lie in a plane with bond angles of120 about C and N.
Model of the zwitterion form of Gly-Gly viewed fromtwo perspectives to show the planarity of the sixatoms of the peptide bond and the preferred s-transgeometry.
Peptide Bond Geometry
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Peptide Bond Geometry
To account for this geometry, Linus Pauling proposed
that a peptide bond is most accurately represented asa hybrid of two contributing structures.
The hybrid has considerable C-N double bondcharacter and rotation about the peptide bond isrestricted.
C
C
N
H
C
C
COO
C N
H
+
-::
:
: : :
Peptide Bond Geometry
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Peptide Bond Geometry
Two conformations are possible for a planar peptide
bond. Virtually all peptide bonds in naturally occurring
proteins studied to date have the s-transconformation.
C
C
O
C N
H
CC
O
C N
H
s-t rans s-cis
Secondary Structure
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Secondary Structure
Secondary structure: The ordered arrangements
(conformations) of amino acids in localizedregions of a polypeptide or protein.
To determine from model building whichconformations would be of greatest stability,Pauling and Corey assumed that:
1. All six atoms of each peptide bond lie in the sameplane and in the s-trans conformation.
2. There is hydrogen bonding between the N-H group ofone peptide bond and a C=O group of another peptidebond as shown in the next screen.
Secondary Structure
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Secondary Structure
Hydrogen bonding between amide groups.
Secondary Structure
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Secondary Structure
On the basis of model building, Pauling and
Corey proposed that two types of secondarystructure should be particularly stable:
-Helix.
Antiparallel -pleated sheet.
-Helix:A type of secondary structure in which asection of polypeptide chain coils into a spiral,most commonly a right-handed spiral.
The -Helix
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The Helix
The polypeptide chain is repeating units of L-alanine.
H bonds (C=OH) parallel to axis of helix
The -Helix
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The Helix
In a section of -helix:
There are 3.6 amino acids per turn of the helix. Each peptide bond is s-transand planar.
N-H groups of all peptide bonds point in the samedirection, which is roughly parallel to the axis of the
helix. C=O groups of all peptide bonds point in the opposite
direction, and also parallel to the axis of the helix.
The C=O group of each peptide bond is hydrogen
bonded to the N-H group of the peptide bond fouramino acid units away from it.
All R- groups point outward from the helix.
The -Helix
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The Helix
An -helix of repeating
units of L-alanine A ball-and-stick model
viewed looking downthe axis of the helix.
A space-filling modelviewed from the side.
-Pleated Sheet
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Pleated Sheet
The antiparallel -pleated sheet consists of
adjacent polypeptide chains running in oppositedirections:
Each peptide bond is planar and has the s-transconformation.
The C=O and N-H groups of peptide bonds fromadjacent chains point toward each other and are in thesame plane so that hydrogen bonding is possiblebetween them.
All R- groups on any one chain alternate, first above,then below the plane of the sheet, etc.
-Pleated Sheet
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Pleated Sheet
-pleated sheet with three polypeptide chains running
in opposite directions (antiparallel).
Tertiary Structure
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Tertiary Structure
Tertiary structure: The three-dimensional
arrangement in space of all atoms in a singlepolypeptide chain.
Disulfide bonds between the side chains of cysteineplay an important role in maintaining 3 structure.
Tertiary Structure
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Tertiary Structure
A ribbon model of myoglobin.
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Quaternary Structure
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Quate a y St uctu e
The quaternary structure of hemoglobin. The -chains
in yellow, the heme ligands in red, and the Fe atoms aswhite spheres.
Quaternary Structure
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Q y
If two polypeptide chains, for example, each have one
hydrophobic patch, each patch can be shielded fromcontact with water if the chains form a dimer.
ProteinNumber of
Subunits
Insulin
Hemoglobin
Alcohol dehydroge nase
Lactate d ehydrogenase
Aldolase
Glutamine sy nthetase
Tobacco mosaic vi rus
protein d isc
6
4
2
4
4
12
17