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Azin Nowrouzi, PhD

TUMS

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Proteins

• Most abundant biological macromolecules.• In all cells and all parts of cells.• Great variety

– Different kinds– Different sizes, and properties– Diversity of biological functions and activities

• 20 precursor molecules are the alphabet of the language of protein structure.

• These precursor molecules are called amino acids.

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What is an amino acid?

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General structure of amino acids

General structure of an amino acid

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Classification of Amino Acid

1. Non polar (Hydrophobic)

2. Polar

3. Charged – Basic– Acidic

Amino acid Classification is based on the properties of the Side Chains or R groups when placed in water, in particular, their polarity or tendency to interact with water at biological pH (near ph 7.0):

I. Neutral amino acids 1. Non polar aliphatic (Gly, Ala, Val, Leu, Ile)

2. Polar, aliphatic (Ser, Thr, Asn, Gln)

3. Aromatic (Phe, Tyr, Trp)

4. Sulfur-containing (Cys, Met)

5. Containing seconcary amino group (pro)

II. Acidic amino acids (Asp, Glu)

III. Basic amino acids (Lys, Arg, His)

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Types of amino acids

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Other types of amino acids

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Amino Acids Abbreviations

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Some facts about amino acids• The first to be discovered was asparagine, in 1806.

– Asparagine was first found in asparagus.

• The last of the 20 to be found, threonine, was not identified until 1938.

• Glutamate was found in wheat gluten.• Tyrosine was first isolated from cheese (its name is

derived from the Greek tyros, “cheese”).• Glycine (Greek glykos,“sweet”) was so named because

of its sweet taste.• Histidine has an imidazole group in its side chain.• Lysine has an amino group in its R.• Arginine has a Guanidine group in its side chain.

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Side chains with unique properties

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• L-threonine is 2S-3R.

Diastereomers

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Disulfide bridge

• Reversible formation of a disulfide bond by the oxidation of two molecules of cysteine.

• Disulfide bonds between Cys residues stabilize the structures of many proteins.

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Properties of Amino Acid Side Chains

The twenty kinds of side chains vary in1. Size & Shape: Large, bulky aromatic to large

aliphatic to small and very flexible CH3 and H

2. Charge (3 Basic; 2 Acidic)

3. Hydrogen-bonding capacity

4. Chemical Reactivity

5. Polarity (7 polar; 8 non-polar)

6. Hydrophobicity

Thus, providing any given protein molecule with functional diversity and versatility.

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Uncommon amino acids in proteins“post-translational modifications”

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Nonprotein amino acidsIn humans, non-protein amino acids also have biologically-important roles. • Glycine, gamma-aminobutyric acid and glutamate are

neurotransmitters and many amino acids are used to synthesize other molecules, for example:

• Tryptophan is a precursor of the neurotransmitter seretonin.• Glycine is a precursor of porphyrins such as heme. • Arginine is a precursor of nitric oxide. • Carnitine, made in the body from the amino acids lysine and

methionine, is used in lipid transport within a cell. • Ornithine and S-adenosylmethionine are precursors of polyamines.• Ornithine and Citrulline are key intermediates (metabolites) in the

biosynthesis of arginine and in the urea cycle. • Homocysteine is an intermediate in S-adenosylmethionine recycling.

Amino acid derivative Use in industry

Aspartame (aspartyl-phenylalanine-1-methyl ester) Low-calorie artificial sweetener.

5-HTP (5-hydroxytryptophan) Treatment for depression and the neurological problems of phenylketonuria.

L-DOPA (L-dihydroxyphenylalanine) Treatment for Parkinsonism.

Monosodium glutamate Food additive that enhances flavor. Confers the taste umami.

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Nutritional importance

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Physical and chemical properties of amino acids

1. Optical properties of amino acids– Stereoisomerism in -amino acids

2. Ionization of amino acids– Titration curves– Isoelectric pH (pI)

3. Chemical reactions of the carboxyl group– Include all the reactions of weak acids

• Salt production with bases• Esters with alcohols• Amides with amines• Decarboxylation

4. Chemical reactions of the amino group– Reaction with dinitrofluorobenzene (DNP-aa)– Amides with acarboxilic acids– Reaction with phenyl isothiocyanate– Reaction with ninhydrin

5. Separation and analysis of amino acids

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C

C

R1

HNH3

+

OO

C

C

R1

H NH3

+

O O

L- and D- configurations are mirror images

-carbon, C, is a chiral center. Mirror plane

L-amino acid D-amino acid

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Stereoisomerism in -amino acids

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Fischer projection formulas

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Different forms of amino acids• When an amino acid is dissolved in

water, it exists in solution as the dipolar ion, or zwitterion (German for “hybrid ion”).

• Substances having this dual nature of acid and base are amphoteric and are often called ampholytes (from “amphoteric electrolytes”).

A zwitterion can act as an acid (proton donor):

A zwitterion can act as a base (proton acceptor):

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Amino acids as diprotic acids• A simple monoamino monocarboxylic -

amino acid, such as alanine, is a diprotic acid when fully protonated—it has two groups, the -COOH group and the -NH3+ group, that can yield protons:

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Titration of an amino acidShown here is the titration curve of 0.1 M glycine at 25 C.

• The ionic species predominating at key points in the titration are shown above the graph.

• The shaded boxes, centered at about pK1 2.34 and pK2 9.60, indicate the regions of greatest buffering power.

Titration Curves Predict the Electric Charge of Amino Acids:Glycine has a net negative charge at any pH above its pI and will thus move toward the positive electrode (the anode) when placed in an electric field. At any pH below its pI, glycine has a net positive charge and will move toward the negative electrode (the cathode).

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Isoelectric pH (pI)• pI= the point where the charges on a zwitterionic molecule

balance• Each amino acid has an isoelectric focusing point (pI).

H3N C C OH

O

H

+H3N C C O

O

H

+ - H2N C C O

O

H

-H H H

pKa1 = 2.34pKa2 = 9.60

Net charge

+1

Net charge

0

Net charge

-1

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Titration curve for glutamate

• There are three ionizable groups and, therefore, three transitions in the titration.

• The pI corresponds to the midpoint between the two transitions that involve the species with no net charge.

• These amino-acids have negative net charge at neutral pH.

Net charge= +1 Net charge= 0 Net charge= -1 Net charge= -2

21 RpKapka

pI

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Titration curve for HistidineNet charge= +2 Net charge= +1 Net charge= 0 Net charge= -1

22 RpKapKa

pI

• There are three ionizable groups and, therefore, three transitions in the titration.

• The pI corresponds to the midpoint between the two transitions that involve the species with no net charge.

• These amino-acids have positive net charge at neutral pH.

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pK1 = 2.18pK2 = 8.95pKR = 10.5

H3N C C

CH2

OH

O

CH2

CH2

CH2

NH3

H

H3N C C

CH2

O

O

CH2

CH2

CH2

NH3

H

H2N C C

CH2

O

O

CH2

CH2

CH2

NH3

H

H2N C C

CH2

O

O

CH2

CH2

CH2

NH2

H

+

+

+ -

+

-

+

-

pI of lysineNet charge

+2

Net charge

+1

Net charge

0

Net charge

-1

pKa1pKa2 pKaR

22 RpKapKa

pI

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COOH

NH3

COOH

NH3

H3C

HN

NH

NH

C NH2

NH

groups pKa

3.5-4.0

9.0-10.8

CH2

12.5

OH

4.3-4.5

CH2

10.1

SH 8.3

10.5

6.0

charge at pH 7.0

-

+

-

+

+

+

0

0

Typical titratable groups on amino acids

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Properties of Non-polar (Hydrophobic) aminoacids

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Properties of Polar (Hydrophilic) amino acids

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Peptides and proteins• Polymers of amino acids.• Two amino acids join covalently through a peptide bond.• Another name for peptide bond is amide bond or linkage.

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water is removed

Peptide bond formation

• There are only 3 known ways to make a peptide bond1. Chemical abiotic synthesis in the laboratory.2. Genetic engineering cloning mechanisms.3. Biologically in cells.• Peptide bonds in proteins are quite stable, with an average half-life

(t1/2) of about 7 years under most intracellular conditions.

This is a condensation reaction.

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Peptides are chains of amino acids

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Chains of amino acids

• Dipeptide: 2 amino acids (aas) joined by 1 peptide bond.

• Tripeptide: 3 aas joined by 2 peptide bonds.• Tetrapeptide: 4 aas joined together by 3 peptide

bonds.• Peptapeptide and so forth…. • Oligopeptide: Few aas joined together by

peptide bonds.• Polypeptide: Many aas joined. Molecular weights

generally below 10000.• Proteins: Many aas joined. Generally have high

molecular weights.

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Nomenclature

• The pentapeptide serylglycyltyrosylalanylleucine, or Ser–Gly–Tyr–Ala–Leu.

• Peptides are named beginning with the amino terminal residue, which by convention is placed at the left.

• The peptide bonds are shaded in yellow; the R groups are in red.

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Ionization behavior of peptides

carboxyl-terminal (C-terminal) residue

amino-terminal (N-terminal) residue

Like free amino acids, peptides have characteristic titration curves and a characteristic isoelectric pH (pI) at which they do not move in an electric field.

Alanylglutamylglycyllysine

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Additional topics

1. Relationship between the activity and size (Mw) of peptides and proteins.

– Small peptides have important biological activities

2. Length of polypeptide chains.– How to calculate the number of amino acids in a

protein.

3. Amino Acid Compositions of polypeptides.

4. Simple and conjugated proteins.

5. Multisubunit proteins.

6. Levels of structure in proteins.

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Peptides have important biological activities

Peptide hormones Oxytocin- a nonapeptide

involved in parturition and lactation.

Vasopressin-maintains water balance Structurally similar, but

different functions Contain disulfide bridges

covalent linkages intramolecular cross-links.

Enkephalins Either of two penta-peptides

with opiate & analgesic activity (involved in pain control).

Occur naturally in brain & have marked affinity for opiate receptors.

• Aspartame low calorie sweetener L-aspartyl-L-phenylalanine

methyl ester.

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Relationship between the activity and size (Mw) of peptides and proteins

Some of vertebrate hormones are small peptides:

• Oxytocin (nine amino acid residues), which is secreted by the posterior pituitary and stimulates uterine contractions.

• Bradykinin (nine residues), which inhibits inflammation of tissues.• Thyrotropin-releasing factor (three residues), which is formed in the

hypothalamus and stimulates the release of another hormone, thyrotropin, from the anterior pituitary gland.

• Some mushroom poisons, such as amanitin.• Many antibiotics.

Slightly larger are small polypeptides and oligopeptides: • The pancreatic hormone insulin, which contains two polypeptide chains,

one having 30 amino acidresidues and the other 21.• Glucagon, another pancreatic hormone, has 29 residues; it opposes the

action of insulin. • Corticotropin is a 39-residue hormone of the anterior pituitary gland that

stimulates the adrenal cortex.

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Insulin• Regulates glucose uptake.

• Diabetes is caused by the failure to produce insulin or the failure to respond to insulin.

• A 51 amino acid protein.• First protein to have its

sequence determined.

• Two chains connected by disulfide bonds.1. alpha chain of 30 aa’s & 2. beta chain of 21 aa

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Length of polypeptide chains• Lengths vary considerably.

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How to calculate the number of amino acids in a protein

• We can calculate the approximate number of amino• acid residues in a simple protein containing no other

chemical constituents by dividing its molecular weight by 110.

• Although the average molecular weight of the 20 common amino acids is about 138, the smaller amino acids predominate in most proteins.

• If we take into account the proportions in which the various amino acids occur in proteins, the average molecular weight of protein amino acids is nearer to 128.

• Because a molecule of water (Mr 18) is removed to create each peptide bond, the average molecular weight of an amino acid residue in a protein is about 128 -18 = 110.

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Polypeptides Have CharacteristicAmino Acid Compositions

• The 20 common amino acids almost never occur in equal amounts in a protein.

• Some amino acids may occur only once or not at all in a given type of protein; others may occur in large numbers.

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Multisubunit proteins

• When two or more polypeptides are associated noncovalently.– Hemoglobin, for example, has four polypeptide

subunits: two identical chains and two identical chains, all four held together by noncovalent interactions.

• A few proteins contain two or more polypeptide chains linked covalently. – For example, the two polypeptide chains of insulin are

linked by disulfide bonds. In such cases, the individual polypeptides are not considered subunits but are commonly referred to simply as chains.

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Levels of structure in proteins

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Simple and conjugated proteins• Some proteins contain chemical groups other than amino acids.

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Spectroscopy of amino acids

• Aromatic amino-acids are strong chomophores in the far-uv.• Only the aromatic amino acids absorb light in the UV region

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Absorbance can be measured by UV-spectrophotometer

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Ninhydrin-detection of amino acids• Complete hydrolysis for 24 hr at 110 oC in 6 M HCl.• Amino acids can be detected on the chromatogram by using

ninhydrin. A solution of ninhydrin is sprayed onto the paper and heated. The amino acids show up as purple spots (proline appears yellow).

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Paper chromatograms

2D chromatogram eluting with a different solvent mixture in each direction.

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Electrophoresis• Electrophoresis is a technique that uses the net charge of peptides

(amino acids) as a basis for separation. A potential difference is applied across a solid material (e.g. paper

for amino acid analysis) permeated by an electrolyte.

Anions migrate to the anode and cations to the cathode. The rate of diffusion is related to the size and net charge. Small highly charged proteins migrate more quickly.

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Isoelectric focusing (IEF)• It is difficult to separate two peptides or proteins of

similar MW if they differ only slightly in their net charge at a given pH.

This problem can be overcome by performing the electrophoresis across a pH gradient – known as isoelectric focusing.

As soon as the IEP is reached the proteins carry zero net charge and sostop migrating.

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Isoelectric focusing (IEF)• This technique separates

proteins according to their isoelectric points.

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1. Proteins larger than 50 aa are first hydrolyzed into shorter peptides.

2. Chemical or enzymatic methods hydrolyze proteins at specific sites.

3. Peptides are separated by chromatography

4. Peptides generated by 2 or more cleavage methods are each sequenced separately.

5. Sequences of individual peptides are overlapped together to deduce the entire protein sequence

Specific cleavage of polypeptides

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Protein Sequencing Example