Chemistry of peptides and proteins · than 50 amino acids are called “proteins”. Peptides and Proteins 33. ... 3 amino acids: glutamic acid ... hydrophobic –CH 3 H 3 C

Chemistry of peptides and proteins

Amino acids

• Provide the monomer units from whichthe long polypeptide chains of proteins aresynthesized

Derived Amino Acids:

Derived and Incorporated in proteins:

• Some amino acids are modified after proteinsynthesis such as hydroxy proline and hydroxy lysinewhich are important component of collagen.

• Gamma carboxylation of glutamic acid residues ofproteins is important for clotting process.

Coagulation (also known as clotting) is the process by which blood changes from a

liquid to a gel, forming a blood clot.

Derived Amino Acids

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Derived and Incorporated in proteins:


Derived but not incorporated in tissue proteins:

e.g.: Ornithine, Citrulline, Homocysteine

No-protein amino acids.

L-Ornithine and citrulline• natural amino acid not found in proteins,• play a role in the urea cycle

Derived Amino Acids


Derived Amino Acids


Homocysteineis biosynthesized from methionine by theremoval of its terminal C methyl group

Derived Amino Acids

Seleno cysteine - cysteine analogue with a selenium- in place of the sulfur-containing thiol group.Selenocysteine is present in several enzymes.

Non standard amino acids

Amino acids

L-amino acids and their derivativesparticipate in cellular functions as diverse as

nerve transmission

and the biosynthesis of

✓ porphyrins,

✓ purines,

✓ pyrimidines,

✓ and urea.

• Serotonin is synthesized from Tryptophan

• Serotonin and melatonin and niacin are synthesized from Tryptophan

(vitamin B3)

Catecholamines, Melanin, thyroid hormone

are synthesized from Tyrosine

Epinephrine (adrenaline)

Melanin, thyroid hormone, catecholamines

are synthesized from Tyrosine

First step Iodination

GABA (neurotransmitter) is synthesized from Glutamic acid

γ-amino butyric acid (GABA)

Nitric oxide, a smooth muscle relaxant is synthesized from Arginine.

Aminoacids are precursors for haem, creatine and glutathione, Porphyrins, purines and pyrimidines.

haem

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Reactions of amino acids

1. Reactions due to amino group

2. Reactions due to carboxyl group

3. Reactions due to side chain

4. Reaction due to both amino and carboxyl groups

Reactions due to amino group

Oxidative deamination-α amino group is removed and corresponding α-keto acid is formed. α-keto acid produced is either converted to glucose or ketone bodies or is completely oxidized.


Transamination-Transfer of an α amino group from an amino acid to an α keto acid to form a new amino acid and a corresponding keto acid.

aspartate + -ketoglutarate oxaloacetate + glutamate


Formation of carbamino compound

• CO2 binds to α amino acid on the globin chain of hemoglobin to form carbamino hemoglobin

• The reaction takes place at alkaline pH and serves as a mechanism for the transfer of Carbon dioxide from the tissues to the lungs by hemoglobin.

Reactions due to carboxyl group

1) Decarboxylation- Amino acids undergo alpha decarboxylation to form corresponding amines. Examples-

Glutamic acid GABAHistidine HistamineTyrosine Tyramine

2) Formation of amide linkage• Non α carboxyl group of an acidic amino acid reacts

with ammonia by condensation reaction to form corresponding amides

Aspartic acid Asparagine Glutamic acid Glutamine


1) Decarboxylation- Amino acids undergo alpha decarboxylation to form corresponding amines.

Glutamic acid GABA

Histidine Histamine

Tyrosine Tyramine

GABA is an inhibitory neurotransmitter whose receptors lower muscle

tone, promote relaxation, diminish anxiety, and stimulate digestion.

Histamine is involved in many allergic reactions.

Tyramine acts as a catecholamine releasing agent.

tyrosine

decarboxylase


2) Formation of amide linkage

• Non α carboxyl group of an acidic amino acid reacts with ammonia by condensation reaction to form corresponding amides

Aspartic acid Asparagine

Glutamic acid Glutamine

Reactions due to side chains

1) Ester formation

• OH containing amino acids e.g. serine, threonine can form esters with phosphoric acid in the formation of phosphoproteins.

Phospho-serine

Proteins are commonly modified at serine, tyrosine and threonine amino acids by adding a phosphate group. Phosphorylation is a common mode of activating or deactivating a protein as a form of regulation.

Reactions due to side chains: Glycoproteins

1) Ester formation • OH group containing amino acid can also form:

Glycosides – by forming O- glycosidic bond with carbohydrate residues.

O-linkage: The oxygen atom in the side chain of serine or threonine amino acids is attached to the sugar

N-linkage: The nitrogen atom in the side chain of Asparagine is attached to the sugar.

Reactions due to side chains: Glycoproteins


2) Reactions due to SH group (Formation of disulphide bonds)

• Cysteine has a sulfhydryl group( SH) group and can form a disulphide (S-S) bond with another cysteine residue.

• The dimer is called Cystine

• Two cysteine residues can connect two polypeptide chains by the formation of interchain disulphide chains.

Formation of disulphide bond

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3)Transmethylation

The methyl group of Methionine can be transferred after activation to an acceptor for the formation of important biological compounds.

4)Reactions due to both amino & carboxyl groups

Formation of peptide bond


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Peptide Bonds Link Amino Acids in Proteins

• Peptide bond - linkage between amino acids is a secondary amide bond

• Formed by condensation of the α-carboxyl of one amino acid with the α-amino of another amino acid (loss of H2O molecule)

Ala-Ser

20 amino acids are commonly found in protein.

These 20 amino acids are linked together through“peptide bond forming peptides and proteins (what’sthe difference?).

- The chains containing less than 50 amino acids arecalled “peptides”, while those containing greaterthan 50 amino acids are called “proteins”.

Peptides and Proteins

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Peptide bond formation:

- Each polypeptide chain starts on the left side by free amino group

of the first amino acid enter in chain formation . It is N- terminus.

- Each polypeptide chain ends on the right side by free COOH group of

the last amino acid and termed (C-terminus).34

Peptides

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Peptides

• Amino acids linked by amide (peptide) bonds

Gly Lys Phe Arg Ser

H2N- -COOH

end Peptide bonds end

Glycyllysylphenylalanylarginylserine

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Resonance structure

of the peptide bond

(a) Peptide bond shown as a

C-N single bond

(b) Peptide bond shown as a

double bond (40%)

(c) Actual structure is a hybrid

of the two resonance

forms. Electrons are

delocalized over three

atoms: O, C, N

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Trans and cis conformations

of a peptide group

• Nearly all peptide groups in proteins are

in the trans conformation

Examples of Peptides:

➢Dipeptide ( 2 amino acids joined by one peptide bond):

Example: Aspartame which acts as sweetening agentbeing used in replacement of cane sugar. It is composedof aspartic acid and phenylalanine.

Cannot be intaken by people suffered fromphenylketonuria (phenylalanine hydroxylase iscompletely or nearly completely deficient, andPhe isn’t metabolised to Tyr)

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Aspartame, an artificial sweetener

• Aspartame is a dipeptide methyl ester

(aspartylphenylalanine methyl ester)

• About 200 times sweeter than table sugar

• Used in diet drinks

• Shouldn’t be used with hot solutions and decomposes

during heating

Asp-Phe-OCH3


➢Tripeptides ( 3 amino acids linked by two peptide bonds).

Example: GSH - glutathione which is formed from3 amino acids: glutamic acid, cysteine and glycine.

It protects against hemolysis of RBC (Red Blood Cell) bybreaking H2O2 which causes cell damage.

Glu-Cys-Gly41


➢nonapeptides (9 amino acids)Examples:Two hormones;oxytocine and vasopressin (ADH) also namedantidiuretic hormone.

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➢Polypeptides:10- 50 amino acids:e.g. Insulin hormone,Chain B and Chain A

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There Are Four Levels of Protein Structure

•Primary structure - amino acid linear sequence

•Secondary structure – the type and the shape of the peptide chain, such as a-helices and b-sheets

•Tertiary structure - describes the shape of the fully folded polypeptide chain in space

•Quaternary structure - arrangement of two or more polypeptide chains into multisubunit molecule

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Primary Structure of Proteins

The particular sequence of amino acids that is the backbone of a peptide chain or protein

• ca

Protein structure:Primary structure:

The primary structure of a protein isits unique sequence of amino acids.

Lysozyme, an enzyme that attacksbacteria, consists of a polypeptidechain of 129 amino acids.

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High orders of Protein structure

A functional protein is not just a polypeptide chain, butone or more polypeptides precisely twisted, folded andcoiled into a molecule of unique shape (conformation).This conformation is essential for some protein function

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Secondary Structure

• Results from hydrogen bond between hydrogen of –

NH group of peptide bond and the –O of C=O

(carbonyl oxygen) of another peptide bond.

• Three-dimensional arrangement of amino acids in a

form of - or β-structured of peptide bonds

• Looks like a coiled “telephone cord” ()

• or nearly fully extended polypeptide chain (β)

According to H-bondingthere are two main forms ofsecondary structure:α-helix: is a spiral structure

resulting from hydrogenbonding between one peptidebond and the fourth one

Secondary Structure

β-sheets

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Secondary Structure

β-sheets: is another form ofsecondary structure in which two ormore polypeptides (or segments ofthe same peptide chain) are linkedtogether by hydrogen bondbetween H- of NH- of one chain andcarbonyl oxygen of adjacent chain(or segment).

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Amino acids

Hydrogen

bond

Alpha helix Β-Pleated sheet

Secondary Structure

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The α Helix Is a Common Protein

Secondary Structure

• The α helix is a common type of secondary

structure in proteins.

• It is the predominant structure in α-keratins.

• In globular proteins, about one-fourth of all

amino acid residues are found in α helices

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Stereo view of right-handed -helix

• All side chains project outward from helix axis

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Secondary Structure – Beta Pleated Sheet

• Polypeptide chains are arranged side by

side

• Hydrogen bonds between chains

• R groups of extend above and below the

sheet

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The adjacent polypeptide chains

in a β pleated sheet can be

either

➢ antiparallel (having the

opposite amino-to-carboxyl

orientation)

➢or parallel (having the same

amino-to-carboxyl polypeptide

orientation).

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b-Sheets (a) parallel, (b) antiparallel

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• Typical of fibrous proteins such

as silk (produced by the larva

of the silkworm moth, to make

cocoons) is almost

pure antiparallel beta pleated

sheet

• elements of beta pleated sheet

are found in many protein

domains.

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Tertiary Structure

• Specific overall shape of a protein

• Cross links between R groups of amino acids in chain

disulfide –S–S– +

ionic –COO– H3N–

H bond C=O HO–

hydrophobic –CH3 H3C–

Is determined by a variety of interactions (bond formation) among R groups and between R groups and the polypeptide backbone

Tertiary Structure

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Tertiary Structure

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The weak interactions include:

➢Hydrogen bonds

among polar side chains

➢Ionic bonds

between charged R groups (basic and acidic amino acids)

➢Hydrophobic interactions

among hydrophobic ( non polar) R groups.

Strong covalent bonds

include disulfide bridges, that form between thesulfhydryl groups (SH) of cysteine monomers,stabilize the structure.

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Formation of cystine (disulfide bridge)

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Tertiary Structure

Amino acids

Hydrogen

bond

Alpha helix Pleated sheet

Polypeptide

(single subunit

of transthyretin)

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•Refers to the organization of subunits in a protein with multiple subunits (2 or more chains)

•Subunits (may be identical or different)

•Subunits are held together by many weak, noncovalent interactions (hydrophobic, electrostatic)

Quaternary Structure

Quaternary structure: two or more polypeptidesubunits held together by non-covalentinteraction like H-bonds, ionic or hydrophobicinteractions.


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• Examples on protein having quaternary structure:

– Collagen is a fibrous protein of three polypeptides (trimeric)that are supercoiled like a rope.

• This provides the structural strength for their role inconnective tissue.


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⅓ of structure is glycine, 10% proline, 10% hydroxyproline and 1% hydroxylysine.

• Examples on protein having quaternary structure:

– Hemoglobin is a globular protein with four polypeptide chains (tetrameric)


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Hemoglobin tetramer

(a) Human oxyhemoglobin (b) Tetramer schematic

• Examples on proteinhaving quaternarystructure:

– Insulin : two polypeptide chains (dimeric) held together primarily by disulfide bonds between cysteine residues


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Conjugated proteins

On hydrolysis, give protein part and non protein part and subclassified into:

1- Phosphoproteins: These are proteins conjugated with phosphate group. Phosphorus is attached to oH group of serine or threonine. e.g. Casein of milk and vitellin of yolk.

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2- Lipoproteins:These are proteins conjugated with lipids.Functions: help lipids to transport in blood

Enter in cell membrane structure helping lipid soluble substances to pass through cell membranes.

3- Glycoproteins: proteins conjugated with sugar (carbohydrate)e.g. – Mucin

- Some hormones such as erythropoeitin- present in cell membrane structure- blood groups.

4- Nucleoproteins: These are basic proteins ( e.g. histones) conjugated with nucleic acid (DNA or RNA).e.g. a- chromosomes: are proteins conjugated with DNA

b- Ribosomes: are proteins conjugated with RNA

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5- Metalloproteins: These are proteins conjugated with metal like iron, copper, zinc.

a- Iron-containing proteins: Iron may present in heme such as in- hemoglobin (Hb)- myoglobin ( protein of skeletal muscles and cardiacmuscle), - cytochromes, - catalase, peroxidases (destroy H2O2) - tryptophan pyrrolase (desrtroy indole ring of tryptophan).

Iron may be present in free state ( not in heme) as in:- Ferritin: Main store of iron in the body. ferritin is present in liver,

spleen and bone marrow.- Hemosidrin: another iron store.- Transferrin: is the iron carrier protein in plasma.

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b- Copper containing proteins: e.g. - Ceruloplasmin which oxidizes ferrous ions into ferric

ions. - Oxidase enzymes such as cytochrome oxidase.

c- Zn containing proteins: e.g. Insulin and carbonic anhydrased- Mg containing proteins:e.g. Kinases and phosphatases.

6-Chromoproteins: These are proteins conjugated withpigment. e.g.

- All proteins containing heme (Hb, myoglobin)- Melanoprotein: e.g proteins of hair which contain melanin.

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Protein Hydrolysis

• Break down of peptide bonds

• Requires acid or base, or enzymes, water and heat

• Gives smaller peptides and amino acids

• Similar to digestion of proteins using enzymes

• Occurs in cells to provide amino acids to synthesize other proteins and tissues

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Hydrolysis of a Dipeptide

H3N CH

CH3

C

O

N

H

CH C

OCH2

OH

OH

+

H3N CH

CH3

COH

O

+ CH C

OCH2

OH

OHH3N

H2O, H+

++

heat

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Denaturation - irreversible coagulation

Disruption of secondary, tertiary and quaternary protein structure by

heat/organics (formaline, detergents)

Break apart H bonds and disrupt hydrophobic attractions

acids/ bases

Break H bonds between polar R groups and

ionic bonds

heavy metal ions

React with S-S bonds to form solids: S-Pb-S

agitation

Stretches chains until bonds break

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Reversible coagulation of proteins – is caused by aqueous solutions

of Na+, K+ and NH4+ salts and diluted alcohols, when proteins

precipitate out of a solution.

After adding water to precipitated proteins, the original protein form

is restored (it is called peptization).

a protein

(a coloid)

a gel

coagulation

peptization

Reversible coagulation

Biuret reaction – detection of peptide bond

All proteins, peptides with the chain length of at least 3 amino acids give a positive result

in this test.

This is a typical reaction for identification of peptide bonds.

Biuret reaction – detection of peptide bond

The Biuret reagent is made of sodium hydroxide (NaOH) and

CuSO4 solution.

The reaction of the cupric ions with the nitrogen atoms

involved in peptide bonds leads to the displacement of the peptide hydrogen atoms

under the alkaline conditions.

Protein purification

a processes intended to isolate one ora few proteins from a complex mixture,usually cells, tissues

Protein purification based on physico-chemical properties

Differences in

• size, shape, and solubility

• binding affinity

• isoelectric point

• charged surface residues

• and biological activity

Protein purification strategies

• Size exclusion chromatography

• Affinity chromatography (Metal binding, Immunoaffinity chromatography)

• Separation based on charge or hydrophobicity

• Electrophoresis

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Size-exclusion chromatography(gel filtration)

The column contains a cross-linked polymer with pores of selected size. Larger proteins migrate faster than smaller ones, because they are too large to enter the pores in the beads.The smaller proteins enter the

pores and and their path through the column is longer.

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Affinity chromatography

separates proteins by their binding specificities. The proteins retained on the column are those that bind specifically to a ligand cross-linked to the beads.

After nonspecific proteins are washed through the column, the bound protein of particular interest is eluted by a solution containing free ligand.

Electrophoretic Separation of Proteins

Proteins are amphoteric compounds

Their net charge therefore is determined by pH of medium in which they are suspended

• In a solution with a pH above its isoelectric point, a protein has a net negative charge and migrates towards anode in an electrical field

• Below its isoelectric point, protein is positively charged and migrates towards cathode

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Electrophoretic Separation of Proteins

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Positively charged (cationic) amino acids are attracted to the negative electrode (the cathode), and negatively charged (anionic) amino acids are attracted to the positive electrode (the anode). An amino acid at its isoelectric point has no net charge, so it does not move.

pH 6 buffer solution

A pH of 6 is more acidic than the isoelectric pH for lysine (9.6),

so lysine is in the cationic form. Aspartic acid has an isoelectricpH of 2.8, so it is in the anionic form.

Electrophoresis of Proteins

– Gel electrophoresis allows for the separation of proteins based on charge, size, and shape.

– Polyacrylamide gel electrophoresis (PAGE).

• Allows for better resolution

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Proteins are usually analyzed by sodium dodecyl sulfate polyacrylamide gel

electrophoresis (SDS-PAGE)

• Proteins are usually denatured in presence of a detergent such as sodium dodecyl sulfate (SDS)

• In denaturing SDS-PAGE separations migration is determined not by intrinsic electrical charge of polypeptide, but by molecular weight

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Proteins

• SDS-PAGE

– Use of sodium dodecyl

sulfate (SDS)

• Denatures proteins into

polypeptide strands

• Gives each polypeptide

strand an overall negative

charge

• Proteins studied are

strictly being separated by

size

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anode

•Proteins studied are strictly

being separated by size

Proteins

• SDS-PAGE– Visualization of proteins in

gel• Coomassie Blue

– Milligram amounts of protein.

• Silver stain

– Microgram amounts of protein.

– Size of unknown bands can be determined from comparison to protein molecular weight standard

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http://www.biomedcentral.com/1472-6750/6/22/figure/F2?highres=y

Proteins

Western blotting combines electrophoretic separation (SDS-PAGE) with

detection using specific antibodies

• for the analysis of the target proteins in a mixture.

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Chemistry of peptides and proteins · than 50 amino acids are called “proteins”. Peptides and Proteins 33. ... 3 amino acids: glutamic acid ... hydrophobic –CH 3 H 3 C