Contentagr.asu.edu.eg/uploads/agr/Lecture_3_201503174901.pdfContent: • Structure ofStructure of...

Content :• Structure of PeptideStructure of Peptide

• Peptide Bond Formation

• Naming Peptides

• Biologically active peptidesBiologically active peptides

• Determination of Peptide Primary Structure

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• peptides are amino acid polymers in which the individual amino acid units calledthe individual amino acid units, called residues, are linked together by amide bonds,

tid b dor peptide bonds.

• The peptide bond is an amide bond• The peptide bond is an amide bond.• Amides are very stable and neutral.

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Functions of peptide bond• It usually found in trans configuration.

• The peptide bond is a partial double bond• The peptide bond is a partial double bond.

• N- partially positive, O- partially negative.

•• Shorthand to read peptides: Shorthand to read peptides:

• The amino acids in a peptide or protein are represented by the 3-letter

or one letter abbreviation.

• This is the chemical

• shorthand to write peptide• shorthand to write peptide.

A peptide bond is a covalent bond formed between the b l f AA d th i f itcarboxyl group of one AA and the amino group of its

next AA with the elimination of one H2O molecule.

Peptides can be extended by adding lti l AA th h lti l tidmultiple AAs through multiple peptide

bonds in a sequential order.dipeptide, tripeptide, oligopeptide, polypeptide

Shorthand to read peptides: Shorthand to read peptides: 3-letter or one letter abbreviation.

This is the chemical shorthand to rite peptide

A id• A peptide • is named with names of all amino acids in the

peptide using a -yl ending for the name • is named using the full amino acid name for the g

amino acid at the C terminal N Terminal C Terminal

A i id ti i ti i tid b d d d i ti f th Amino acids participating in peptide bonds are named as derivatives of the carboxyl terminal amino acid residuee.g. NH2-lys-leu-tyr-gln-COOH is called lysylleucyltyrosylglutamine.

Write the name of the following tetrapeptide using amino acid names and three-letter abbreviations.

CH3 S

CH3

CH CH3

3

O O O OCH CH2

CH2

CH2

SH

CH3

H3N CH C N

H

CH C N

H

CH C N

H

CH C O-223

٩

• Draw the structural formula of the following peptide.The pentapeptide “ serylglycyltyrosylalanylleucine

(S Gl T Al L )(Ser–Gly–Tyr–Ala–Leu)

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Learning CheckLearning Check Draw the structural formula of each of the following Draw the structural formula of each of the following

peptides.A.Methionylaspartic acidB.AlanyltryptophanC. MethionylglutaminyllysineD. HistidylglycylglutamylalanineD. Histidylglycylglutamylalanine

١٢

Biologically active peptidesPeptides may act as hormones such as: Peptides may act as hormones such as:

–Vasopressin and oxytocin – hormones released by p y ypituitary gland.

–Both have disulfide bridges.g–Vasopressin (antidiuretic hormone) decreases urine

formation.–Oxytocin causes uterine contractions.

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Peptides may act as hormones such as :Peptides may act as hormones such as :Peptides may act as hormones such as :Peptides may act as hormones such as :Adrenocorticotropic hormone – released by

pituitary glandpituitary glandHas no disulfide bridgesRegulates the production of steroids by theRegulates the production of steroids by the

adrenal gland

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Glutathione (GSHGlutathione (GSH L- glutamylcysteinylglycine

Glutathione is a tripeptide composed Glutathione is a tripeptide composed of gamma glutamate, cystein, glycine.

Reduced glutathione (GSH) maintains Reduced glutathione (GSH) maintains the normal reduced state of the cell.

.عندما يكون مؤكسدا GSSGعندما يكون مختزال، ويرمز له GSHويرمز له بالرمز •إنزيمي• كمرافق أكسدة،Coenzymeويعمل ضررAntioxidantومضاد من الخاليا لحماية

•

ي زي ق إ ر ل رر Antioxidantو Coenzymeوي ن ي ي .Free radicalsالجذور الحرة

.ويعد الجلوتاثيون ھاما لسالمة خاليا الدم الحمراء وعمل البروتينات واألغشية الدھنية وغيرھا•

H2O2 2GSH NADP+

GSH

H2O2 2GSH NADP

GSH can be regenerated from

peroxidase GSH reductase

regenerated from GSSG by the enzyme glutathione reductase (GSR)

2H2O GSSG NADPH+H+ ( )

As a reductant to protect nucleic acids and proteinsAs a reductant to protect nucleic acids and proteins from toxin by discharging free radical or H2O2

InsulinInsulin consists of two polypeptide chains,

A and B, held together by two disulfide bonds.

The A chain has 21 residues and the B chain has 30 residuesThe A chain has 21 residues and the B chain has 30 residues.

Insulin is a peptide hormone produced by beta cells in the pancreas Insulin is a peptide hormone produced by beta cells in the pancreas. It regulates the metabolism of carbohydrates and fats by promoting the

absorption of glucose from the blood to skeletal muscles and fat tissue and by i f t t b t d th th d f

١٧

causing fat to be stored rather than used for energy. Insulin also inhibits the production of glucose by the liver.

Q :Choose the ONE correct answer1. Which one of the following statements concerning the peptide1. Which one of the following statements concerning the peptide shown below is correct?        Gly‐Cys‐Glu‐Ser‐Asp‐Arg‐CysA. The peptide contains glutamine.B. The peptide contains a side chain with a secondary amino group.C. The peptide contains a majority of amino acids with side chains h ld b i i l h d H 7that would be positively charged at pH 7.

D. The peptide is able to form an internal disulfide bond.

Peptides Can Be Distinguished by Their Ionization Behavior (Alanyl‐glutamyl‐glycyl‐lysine)

• The acid‐base behavior of a peptide can be predicted from its free ‐amino and ‐carboxyl groups as well as the nature and number of itsthe nature and number of its ionizable R groups.

• Peptides have characteristic• Peptides have characteristic titration curves and a characteristic isoelectric pH p(pI) at which they do not move in an electric field.

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Calculate the net charge of a peptideor protein at a given pH

A b hi l i l i i h id• At best, this value is only an estimate, since the sidechains of polymerized amino acids do not behave as th d i f i idthey do in free amino acids.

• This is because of the electronic effects of the peptide bond and other functional groups that may be broughtbond and other functional groups that may be brought into proximity when the polypeptide chain folds into a three‐dimensional shape.three dimensional shape.

• The chemical properties of a side chain’s immediate neighbors, its microenvironment, may alter its polarity, g , , y p y,thereby altering its tendency to lose or accept a proton.

• Estimate the net charge of the polypeptide chain below at physiological pH (7.4) and at pH 5.0

Ala–Arg–Val–His–Asp–GlnSOLUTION

• The polypeptide contains the following ionizable groups, whose pK values are listed in Table 4‐1: the N‐terminus (pK 5 9.0), Arg (pK 5 12.5), His (pK 5 6.0),Asp (pK 5 3.9), and the C‐terminus (pK 5 3.5).), p (p ), (p )

• At pH 7.4, the groups whose pK values are less than 7.4 are mostly deprotonated, and the groups with pK values greater than 7.4 are mostly protonated The polypeptide therefore has a net charge of 0 Group Chargeprotonated. The polypeptide therefore has a net charge of 0:Group Charge

PRACTICE PROBLEMSPRACTICE PROBLEMS

• 1. Estimate the net charge of a Glu–Tyr dipeptide at pH 6.0.• 2. Estimate the net charge of an Asp–Asp–Asp tripeptide at pH 

7 07.0.• 3. Estimate the net charge of a His–Lys–Glu tripeptide at pH 8.0.

Determination of Peptide Primary Structurey

What amino acids are present?

How much of each is present?p

In what sequence do the amino acids occur?

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• The primary structure of a protein is the particular sequence of amino acids p q

• The backbone of a peptide chain or protein

• The chains containing less than 50 aminoThe chains containing less than 50 amino acids are called “peptides”, while those containing greater than 50 amino acids arecontaining greater than 50 amino acids are called “proteins”.

• proteins exhibit different behaviors under given laboratory conditions.

• These differences can be exploited to purify a protein, that is, to isolate it from a mixture containing other gmolecules

The first step in determining the sequence of amino acids ina peptide or protein is to cleave the disulfide bridgesa peptide or protein is to cleave the disulfide bridges

The primary structure typically isThe primary structure typically is detemined in several steps

1. Amino Acid Analysis: determine what amino acids are present and their molar ratios.

2. Partial Hydrolysis of Peptides: cleave the peptide into smaller fragments, and determine the amino acid composition of these smaller fragmentsacid composition of these smaller fragments.

3. C- and N-Terminus?: identify the N-terminus and C-terminus in the parent peptide and in each p p pfragment.

4. Sequence Analysis: organize the information so that the sequences of small fragments can be overlapped to reveal the full sequence.

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Step 1: Amino Acid Analysis•Acid-hydrolysis of the peptide (6 M HCl, 24 hr) gives a mixture of amino acidsgives a mixture of amino acids.•The mixture is separated by ion-exchange chromatography which depends on the differenceschromatography, which depends on the differences in pI among the various amino acids.•Amino Acid are measured by reacting them with aAmino Acid are measured by reacting them with a compound called ninhydrin. Alpha Amino acids will be given an intense blue color while imino acids g(proline, hydroproline) will be given a yellow color. •The concentration of a single amino acid is gproportional to the light absorbance of the solution after adding ninhydrin.. •sequence information lost upon hydrolysis

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Total Hydrolysis: conversion of a peptide y y p pinto a mixture of its component amino acids

OHHH O H OHOHHOHHH H O

F M

C C

CH2OH

NH3N C C

CH3 CH2CH2SCH3

CC ONC C

H

NC C

CH(CH3)2

NN

CH2

CC

A F V MG

H3O, heat(total hydrolysis)

S

(total hydrolysis)

OHH O H O OH OH H O

S

C CO

OH

CH2OH

NH3N C CO

H O

CH3

N

CH2

H O

COC C CO

CH(CH3)2

N C CO

H

NCH2CH2SCH3

COCNH3H3H3 H3 H3+ ++ + +

A F V G MS

)

A F V G M

A, S, F, V, G, and M(equimolar mixture of

S G A V M F29Ion Exchange Chromatogram:

Step 2: Partial Hydrolysis of Peptides and Proteins

The enzymes that catalyze the hydrolysis ofThe enzymes that catalyze the hydrolysis of peptide bonds are called peptidases,

proteases or proteolytic enzymesproteases, or proteolytic enzymes.

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• Longer polypeptide chains are broken into shorter g p yp pones for analysis by specifically cleaving them with enzymes that cleave at specific points.

• Some examples are :

Enzyme Cleavage PointEnzyme Cleavage Point

Trypsin Lys, Arg (C)

Chymotrypsin Phe,Trp, Typ (C)

Pepsin Phe, Trp, Tyr (N)

Cyanogen bromide Met (C)

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T iTrypsinTrypsin is selective for cleaving the peptide bond Trypsin is selective for cleaving the peptide bond to the carboxyl group of lysine or arginine.to the carboxyl group of lysine or arginine.

OO OOOO

NNHCHCHCHC

R'R'

NNHCHCHCHC

R"R"

NNHCHCHCHC

RR R'R' R"R"RR

lysine or argininelysine or arginine

Ch t iChymotrypsinChymotrypsin is selective for cleaving the peptideChymotrypsin is selective for cleaving the peptidebond to the carboxyl group of amino acids withbond to the carboxyl group of amino acids withan aromatic side chain.an aromatic side chain.

OO OOOO

NNHCHCHCHC

R'R'

NNHCHCHCHC

R"R"

NNHCHCHCHC

RR R'R' R"R"RR

phenylalanine, tyrosine, phenylalanine, tyrosine, tryptophantryptophan

C b tidCarboxypeptidase

Carboxypeptidase is selective for cleavingCarboxypeptidase is selective for cleavingthe peptide bond to the Cthe peptide bond to the C terminal amino acidterminal amino acidthe peptide bond to the Cthe peptide bond to the C--terminal amino acid.terminal amino acid.

OO++

OOOO

proteinproteinHH33NNCHCCHC++

NNHCHCOHCHCO––

CC

RR RR

Carboxypeptidase:Carboxypeptidase:

C C

OHH

NH N C C

H O H O

CC ON

H

C C

OH

N

H

C C

OH

N

HH

N

H O

CC

A F V MG

C C

C H 2 O H

NH 3N C C

C H 3 C H 2C H 2 S C H 3

CC ONC C

H

NC C

C H (C H 3 )2

NN

C H 2

CC

S

H O

C arbox ype pt ida se

OHHH O OHHOHHH H O

C H 2 C H 2 S C H 3

C OCNH 3+

M

C C

C H 2 O H

NH 3 N C C

C H 3

C C O

H

NC C

C H (C H 3)2

NN

C H 2

CC

OH

C arbox ype pt ida se

OHHH O OHHH H O

C C O

OH

H

NH 3

G

C C

OH

C H 2 O H

H

NH 3 N C C

H O

C H 3

C C O

OH

C H (C H 3)2

N

HH

N

C H 2

H O

CC +

G

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Peptide Digestion

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الببتيديةالسلسلةطرففيالموجودأألمينيالحامضلتشخيصالتفاعلھذايستعمل• الببتيدية السلسلة طرففيالموجودأألمينيالحامضلتشخيصالتفاعلھذايستعمل•)النتروجينية النھاية(

FDNBFDNBالمركبھذايتفاعل• ))1-fluro-2,4- Dinitrobenzene(في ل بي FDNBFDNBر ))1 fluro 2,4 Dinitrobenzene(ي من النتروجينية نھاية في األول األميني الحامض مع المعتدلة القاعدية المحاليل اللون أصفر مركب مكونا الببتيدية السلسلة

Chromatography بواسطة به المرتبط األميني الحامض يشخص• ويعتبر حر بشكل الببتيدية السلسلة من االمينية االحماض تتحرر التفاعل ھذا في •

حر بشكل األمينية االحماضبتحرير وذلك الببتيدية للسلسلة مدمرا التفاعل ھذاNONO22

FFOO22NN39

OO22NN

Sanger’s Reagent( N-terminal Amino Acid Analysis)

NO2 NO2 R OR O+

H+ + HF

O2N F O2N NH CH CH3N CH C+

NO

DNFB(2,4 Dinitrofluorobenzene)

H+

O2N NH

NO2

CH

R

COOH + FreeAmino Acids

DNP-amino acid(Dinitrophenyl-amino acid)

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Carboxypeptidase:Carboxypeptidase:

C C

OHH

NH N C C

H O H O

CC ON

H

C C

OH

N

H

C C

OH

N

HH

N

H O

CC

A F V MG

C C

C H 2 O H

NH 3N C C

C H 3 C H 2C H 2 S C H 3

CC ONC C

H

NC C

C H (C H 3 )2

NN

C H 2

CC

S

H O

C arbox ype pt ida se

OHHH O OHHOHHH H O

C H 2 C H 2 S C H 3

C OCNH 3+

M

C C

C H 2 O H

NH 3 N C C

C H 3

C C O

H

NC C

C H (C H 3)2

NN

C H 2

CC

OH

C arbox ype pt ida se

OHHH O OHHH H O

C C O

OH

H

NH 3

G

C C

OH

C H 2 O H

H

NH 3 N C C

H O

C H 3

C C O

OH

C H (C H 3)2

N

HH

N

C H 2

H O

CC +

G

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Edman degradationg

1. Labeling the N-terminal residue with a gfluorophore.

2 Cleaving the labeled residue without2. Cleaving the labeled residue without breaking the peptide bonds of the rest part of the peptide.

3 Determining the N-terminal residue with3. Determining the N-terminal residue with chromatography.

4. Repeating the same procedure to the rest peptide until the whole sequence is pept de u t t e o e seque ce sdetermined.

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Determination of protein sequenceEd d d tiEdman degradation

• Based on the use of phenyl• Based on the use of phenylisothiocyanate(PTC; Edman’s reagent);

• PTC reacts with and labels the amino acid at the N-terminus ofamino acid at the N terminus of the peptide;

Th PTC l b l d i id i• The PTC-labeled amino acid isreleased from the peptide and identified by chromatography;y g p y

• Cycles of labeling and release allow one to determine theallow one to determine the sequence of the peptide.

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Determination of protein sequenceEdman degradation

Identifyy

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Putting it all together!Putting it all together!• Suppose an unknown hexapeptide gave

“tagged” A (alanine) upon treatment with Sanger’s reagent, and upon treatment with carboxypeptidase, the first amino acid released was M (methionine) followed by G (glycine)

• Partial hydrolysis gave the following identifiable tripeptides: V-G-M, A-S-F, and S-F-V. What is p p , ,the 1º structure of the hexapeptide?A S F V G M

V G MA S F V G M

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A S F V G MA S F V G MA S F V G M

– Complete Sequence Analysis– The Sanger and Edman methods of analysis apply to short

l tid ( t b t 60 i id id bpolypeptide sequences (up to about 60 amino acid residues by Edman degradation)

– For large proteins and polypeptides, the sample is subjected to g p p yp p , p jpartial hydrolysis with dilute acid to give a random assortment of shorter polypeptides which are then analyzed

The smaller polypeptides are sequenced and regions of– The smaller polypeptides are sequenced, and regions of overlap among them allow the entire polypeptide to be sequenced

• Example: A pentapeptide is known to contain the following amino acids:

– Using DNFB and carboxypeptidase, the N-terminal and C-terminal amino acids are identifie

– The pentapeptide is subjected to partial hydrolysis and the following dipeptides are obtained

– The amino acid sequence of the pentapeptide must be46

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• Determine the sequence of a hexapeptide based on the following data Note: when the sequence is not known afollowing data. Note: when the sequence is not known, a comma separates the amino acids

• Amino acid composition: (2R A S V Y)• Amino acid composition: (2R,A,S,V,Y) • N-terminal analysis of the hexapeptide: A • Trypsin digestion: (R A V) and (R S Y)• Trypsin digestion: (R,A,V) and (R,S,Y) • Carboxypeptidase A digestion: No digestion • Chymotrypsin digestion: (A R V Y) and (R S)• Chymotrypsin digestion: (A,R,V,Y) and (R,S)

R= arg A =Ala S= Ser V=Val Y=Tyr

The sequence is : AVRYSR

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Peptide primary structure problemPeptide primary structure problem• An unknown octapeptide gives the following upon total hydrolysis:

A(2), C, D, G, L, M, S• Reaction of the octapeptide with Sanger’s reagent followed by total

hydrolysis gives “labeled” leucine (L). • Carboxypeptidase treatment of the octapeptide gives initially a high

concentration of alanine (A) followed by glycine (G) and then serine (S)concentration of alanine (A), followed by glycine (G) and then serine (S). • Leucine aminopeptidase treatment of the octapeptide gives initially a high

concentration of leucine (L), followed by aspartic acid (D) then cysteine (C).• Partial hydrolysis of the octapeptide gives the following identifiable y y p p g g

fragments• D – C – M, A – S, C – M – A, S – G – A, and L – D• Write the correct primary structure (using one-letter abbreviations and

following the usual convention of listing the N terminal amino acid on thefollowing the usual convention of listing the N-terminal amino acid on the left).

D – C – MA S

D C MC – M – A

S – G – AA – S

L – D

L AGSD CN-terminal aa C-terminal aa

M A

Determination of protein sequence1. Enzyme mapping – example 2

The following data were obtained after treating an octopeptide with The following data were obtained after treating an octopeptide with the following reagents:

– HCl 6M: Ala, Gly2, Lys, Met, Ser, Thr, Tyr

– Chymotrypsin: 2 peptides wereobtained:

• Peptide 5: Gly Tyr– CNBr: 2 peptides were obtained:

• Peptide 1: Ala, Gly, Lys, Thr• Peptide 2: Gly, Met, Ser, Tyr

Peptide 5: Gly, Tyr• Peptide 6: Ala, Gly, Lys, Met, Ser,

Thr

FDNB i ld Glp y, , , y

– Trypsin: 2 peptides were obtained:• Peptide 3: Ala, Gly

P tid 4 Gl L M t S Th

– FDNB: yields Gly

– Carboxypeptidase A: yields Gly• Peptide 4: Gly, Lys, Met, Ser, Thr,

Tyr

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What is the sequence of this peptide?

THE ENDTHE END

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