27 amino acids, proteins

7/31/2019 27 Amino Acids, Proteins

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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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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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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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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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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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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.



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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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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.



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The solid support used in the Merrifield solid

phase synthesis.


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Merrifield synthesized the

enzyme ribonuclease, aprotein containing 124 aminoacids

Peptide Bond Geometry


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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.



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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

+

-::

:

: : :



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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

27 amino acids, proteins

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