HOLY SPIRIT UIVERSITY OF KASLIK

Faculty of Arts and Sciences

Structural biochemistry
BCH320
Chapter III: Amino acids and
Proteins

Dr. Tania Bitar

Ref: Garrett&Grisham Biochemistry, 6th Edition

 Amino acids
Structure and properties

All objects have mirror images, and amino

acids exist in mirror-image forms.
Only the L-isomers of amino acids occur
commonly in nature.
 Amino acids
Structure and properties

• Amino acids are the building blocks of proteins.

• The stunning diversity of the thousands of proteins found in nature arises from the intrinsic properties of only 20 commonly
occurring amino acids.
 Amino acids
Structure and properties
● Typical Amino Acids Contain a Central Tetrahedral Carbon Atom
• Central to the structure of an amino acid is the carbon (Ca), which is covalently linked to both the amino group and the
carboxyl group. Also bonded to this a-carbon are a hydrogen and a variable side chain.
• In neutral solution (pH 7), the carboxyl group exists as –COO- and the amino group as -NH3+.
• The molecule is called zwitterion.
• Amino acids are also chiral molecules. The a-carbon is said to be asymmetric.
• The two possible configurations for the a-carbon constitute non identical mirror image isomers or enantiomers.
 Amino acids
Structure and properties
● Amino Acids Can Join via Peptide Bonds

• Two identifying chemical groups: the amino (-NH3+) and

carboxyl (-COO-) groups.
• Two amino acids can react with loss of a water molecule to form
a covalent bond. The bond joining the two amino acids is called a
peptide bond.
• The remarkable properties of proteins depend on the unique
properties and chemical diversity of the 20 common amino acids
found in proteins.
 Amino acids
Structure and properties
● There Are 20 Common Amino Acids

• There are several ways to classify the common amino acids.

• The most useful of these classifications is based on the polarity of the side chains.
(1) nonpolar or hydrophobic amino acids,
(2) neutral (uncharged) but polar amino acids,
(3) acidic amino acids (which have a net negative charge at pH 7.0)
(4) basic amino acids (which have a net positive charge at neutral pH).
 Amino acids
Structure and properties
● There Are 20 Common Amino Acids
 Amino acids
Structure and properties
● There Are 20 Common Amino Acids
 Amino acids
Structure and properties
● There Are 20 Common Amino Acids
 Amino acids
Structure and properties
● There Are 20 Common Amino Acids
 Amino acids
Structure and properties
● Several Amino Acids Occur Only Rarely in Proteins

• There are several amino acids that occur only rarely in proteins and are produced by modifications of one of the 20 amino aci

➢ Hydroxylysine and hydroxyproline are

found in connective-tissue proteins;
➢ carboxy-glutamate is found in blood-clotting
proteins;
➢ pyroglutamate is found in bacteriorhodopsin
 Amino acids
Structure and properties
● Several Amino Acids Occur Only Rarely in Proteins

➢ Epinephrine, histamine, and serotonin, although not amino acids, are derived from and closely related to amino acids.
 Amino acids
Structure and properties
● Properties of Amino Acids

1. Stereoisomerism:

• The α carbon atom of all amino acids with the exception of glycine is asymmetrical since it is bonded
to 4 different atoms: -H, -NH2, -COOH and –R (lateral chain)

• There exist 2 stereoisomers of amino acids: D and L amino acids based on the location of the NH2
group

• The carbon atom is considered a chiral center and two stereoisomers are considered as enantiomers

13
 Amino acids
Structure and properties
● Properties of Amino Acids
1. Stereoisomerism:

The amino acids present in natural proteins adopt

predominantly the L -configuration however some
exceptions exist
D-amino acids predominates in the polypeptides of the
bacterial wall and in bacterial antibiotics
D-amino acids predominates in polypeptides synthesized
at the ribosomal level is eukaryotes and prokaryotes
14
 Amino acids
Structure and properties
● Properties of Amino Acids

1. Ionization of Amino Acids:

• All amino acids possess at least two ionisable groups: the carboxylic acid group and the amine group

• Amino acids are amphoteric compounds: they can exist at different forms of ionisation based on the pH

Acidic pH: R-NH2 + H+ R-NH3+

Basic pH: R-COOH R-COO- + H+

15
 Amino acids
Structure and properties
● Properties of Amino Acids

At pH 7, the COOH group is ionized to COO-, and the NH2 group is ionized to NH3+ at pH 7 an amino acid is
dipolar (an electrically neutral molecule carrying both a positive and a negative charge)

COO-: carboxylate ion

NH3+: Ammonia

16
 Amino acids
Structure and properties
● Properties of Amino Acids

1. Ionization of Amino Acids:

• At acidic pH ( excess H+), the amine group will gain a proton to become a cation

• At basic pH (excess OH-), the carboxylic acid group will lose a proton to become an anion

• pHi: isoelectric pH, the pH at which a particular molecule carries no net electrical charge (mi-distance between
2 pka)

Zwitterion
Acidic pH Basic pH
pHi 17
pH < pHi pH > pHi
 Amino acids
Structure and properties
● Properties of Amino Acids

1. Ionization of Amino Acids:

• Zwitterions are simultaneously electrically charged and electrically neutral. They contain positive and negative
charges, but the net charge on the molecule is zero.
• pHi=(pKCOOH+ pKNH2)/2

• At pHi the amino acid is mostly dissociated and its net charge is 0 zwitterion ion

18
 Amino acids
Structure and properties
An amino acid is characterized by two pK with the following values: 2.32 and 9.68. Is there in aqueous solution at pH
6 a major form of this amino acid? If yes which one?

19
 Amino acids
Structure and properties
● Properties of Amino Acids

1. Ionization of Amino Acids:

• Since the αcarboxylic acid and αamine functional groups will be engaged in peptide bonds in polypeptide chains,
the charge of proteins will depend mainly on the charge of the lateral chains of basic and acidic amino
acids

20
 Amino acids
Structure and properties
● Chemical Properties of Amino Acids
O O O
General reaction for identification of aa: C COOH R C
R
OH H
• Ninhydrin Reaction
OH CO2
O O
ninhydrin H
+ NH3
OH
+
R C COOH O
H ninhydrin
NH2
aa
3 H2O
O O
N Purple color

O O 21
 Amino acids
Structure and properties
● Chemical Properties of Amino Acids
Specific properties for certain aa

O O
H2N CH C H2N CH C

CH2 OH OH
CH2
Ser
O
O
H

OH H2O
HO P O

HO P O
OH

OH Phosphoserine 22
Phosphate
 Amino acids
Structure and properties
● Chemical Properties of Amino Acids
Specific properties for certain aa

O
H2N CH C
CH3 CH OH
O
H2N CH C
Thr O
OH
CH3 CH
H

OH O

HO P O
H2O HO P O
Phosphothreonine
OH
23
OH
 Amino acids
Structure and properties
● Principal reactions of Amino acids

24
 Amino acids
Structure and properties
● Principal reactions of Amino acids

Reactions due to the presence of –COOH

1- Amidation: Amide: -COOH of an a.a + -NH2 of another molecule with the departure of a molecule of H2O

2- Decarboxylation: catalyzed by a decarboxylase with pyridoxal phosphate coenzyme (PLP) => production of
biologically important amines (histidine -> histamine, serine -> ethanolamine) or precursors of other molecules
(aspartate -> CoA)

25
 Amino acids
Structure and properties
● Principal reactions of Amino acids

Reactions due to the presence of –NH2

1- Amidation (already explained)

2- Transamination catalyzed by aminotransferase (AT) (transaminase): reversible transfer reaction of –NH2

between an a.a and an α-keto acid

The a.a is transformed into α-keto acid

The α-keto acid acceptor of –NH2 is transformed into a.a (the α-keto acid acceptor is almost always α-
ketoglutarate transformed into glutamate)

26
 Amino acids
Structure and properties
● Principal reactions of Amino acids

O aminotransferase O
R C C R C C
H
OH OH
NH2 PLP O

aa a -Keto acid

a -KG Glu
O
O HOOC-CH2-CH2 C C
HOOC-CH2-CH2 C C H
NH2 OH
O OH
27
 Amino acids
Structure and properties
● Principal reactions of Amino acids
Deamination:

Oxidative deamination:

-catalyzed by a dehydrogenase

-transforms an a.a into the corresponding α-keto acid and takes place in 2 steps:

-oxidation of a.a to α-imino acid (reduction of NAD(P)+ to NAD(P)H,H+)

-hydrolysis of unstable α-imino acid to α-keto acid with release of NH3 (NH3 +H2O -> NH4OH, strong base)

28
 Amino acids
Structure and properties
● Principal reactions of Amino acids
O Amino-acide O
R C C R C
oxydase C
H
OH OH
NH3+ NH2+
PLP
Imino-acide
Acide a -aminé NAD NADH,H+
H2O

NH3.

O
R C C
Acide a -cétonique OH 29
 Proteins
Structure and properties
➢ Definition:

• Proteins are polymers of amino acids linked by an amide bond called “peptide bond”

• The peptide bond is formed between the αcarboxylic group of the first amino acid and the αamine group of the
second

• Peptide = (a.a)n

• Dipeptide: 2 a.a

• Oligopeptide: 2< n <10 Peptide bond

• Polypeptide: n >10<100

• Protein: n >100

Free N-extremity Free C-extremity

 Proteins
Structure and properties
➢ Definition:

• All proteins are constituted of 20 amino acids

• The combination of these amino acids in proteins is unlimited: 20n for a protein made up of n amino acids

• Following their synthesis proteins may be linked to non-protein molecules such as carbohydrates, which increases their
structural diversity
 Proteins
Structure and properties
➢ Biological functions of proteins:

• Proteins are highly important biological molecules:

➢ Quantitative: they constitute more than half of the net

weight of cells

➢ Qualitative: they are involved in almost all of the

cellular functions
 Proteins
Structure and properties
➢ Classification of proteins:

• Proteins are classified based on their composition into:

• Simple proteins: composed of amino acids ONLY
• Conjugated proteins: composed of proteins and non-proteins molecules called prosthetic groups, which
may be carbohydrates, lipids, nucleic acids…

• Proteins are classified based on their form into:

• Globular Proteins
• Fibrous Proteins
• Membrane Proteins
 Proteins
Structure and properties
➢ Classification of proteins:
➢ Globular Proteins:
• spheroidal proteins
• Soluble in water
• Comprise hormones, enzymes and antibodies
➢ Fibrous Proteins
• Filiform proteins (filamentous, thread-like)
• Insoluble in water
• Perform structural and protective functions (keratin and collagen)
• Membrane proteins
• Found in association with the various membrane systems of cells.
 Proteins
Structure and properties
➢ Structure of proteins:

I. Primary
II. Secondary
III. Tertiary
IV. Quaternary
 Proteins
Structure and properties
➢ Primary structure of proteins:

• The primary structure of proteins corresponds to the sequence of the constituting amino acids from the N-terminal to
the C-terminal linked by peptide bonds

• The primary structure of a protein is determined by the gene encoding this protein

• An alteration in the coding part of this gene results in the modification of the primary structure of the protein
perturbation in the secondary and tertiary structures of the protein
 Proteins
Structure and properties
➢ Secondary structure of proteins:

The secondary structure of a protein corresponds to the organization of its peptide bonds in space
This organization is maintained and stabilized by hydrogen bonds between the C = O and N – H groups of two
peptide bonds
The secondary structure confers an organized aspect to proteins
The secondary structure of proteins may adopt 3 different forms:
➢ α-helix
➢ β-Pleated sheets Hydrogen
➢ β-turns Bonds
 Proteins
Structure and properties
➢ Secondary structure of proteins:

α-Helix Structure:

• This structure is stabilized by intra-chain hydrogen bonds that form between the
oxygen atom of the C = O group of an amino acid and the N-H group of the next fifth
amino acid in the chain.

• Hence there exists 3 amino acid residues between the two peptide bonds

• The hydrogen bonds are located at the interior of the helix whereas the amino acids
are located at the exterior

• The Keratin of our hair in a αHelix that forms elongated fiber.

 Proteins
Structure and properties
➢ Secondary structure of proteins:

βSheet Structure:
• β-sheets consist from βstrands connected laterally by at least
two or three backbone hydrogen bonds, forming a generally
twisted, pleated sheet

➢ Parallel βSheets the two strands are in the same directions

➢ Anti-parallel βSheets the two strands are in opposite

directions – more stable
 Proteins
Structure and properties
➢ Secondary structure of proteins:

Βturns
Structure:
Each β-turn consists of four amino acid residues
(labelled i, i+1, i+2, i+3)

This structure is maintained by the formation of

hydrogen bond between the C = O group of the Hydrogen bonds
first amino acid and the N – H group of the
fourth amino acid

This structure allows the folding of the protein

chain
 Proteins
Structure and properties
➢ Tertiary structure of proteins:

• The tertiary structure is stabilized by the formation of bonds between the lateral chains of the amino acids
residues, which were distant in their primary structure structure but became close following the folding of the
polypeptide chain
• The tertiary structure gives the protein its native form
• It is essentially a picture of what the shape of the entire protein actually looks like
• Every protein spontaneously adopts a unique tertiary structure
 Proteins
Structure and properties
➢ Tertiary structure of proteins:

➢ Covalent bonds:
• Disulfur bonds, between two cysteine residues that were distant in the primary structure but became
close following the folding of the chain
Disulfide
bonds
➢ Non-covalent bonds: Hydrophobic
• Hydrophobic bonds interactions

• Hydrogen bonds
Ionic bonds (Salt
• Ionic bonds bridges)
• Van der waals bonds Hydrogen
bonds
 Proteins
Structure and properties
➢ Quaternary structure of proteins:

• Many proteins consist of two or more interacting polypeptide chains of

characteristic tertiary structure, each of which is commonly referred to as a
subunit of the protein.
• Subunit organization constitutes another level in the hierarchy of protein
structure, defined as the protein’s quaternary (4°) structure

ATTENTION:
Not all proteins necessarily possess a quaternary structure
 Proteins
Structure and properties
 Proteins
Structure and properties
➢ Proteins: Forms

Fibrous proteins

- Keratins

• Secondary structure = α helix

• Hard keratins or scleroproteins: role in protecting the external surfaces of animals (nails, hair and hair, beaks and
feathers, scales and claws; in a word, the appendages)

• Soft keratins such as cytokeratins that structure epithelial cells

 Proteins
Structure and properties
➢ Proteins: Forms

Fibrous proteins

- Keratins are:

• Extensible: a helix can be stretched which breaks the H bonds; released, it resumes its compact state and the H
bonds reform

• Insoluble in water: the –R side chain, mostly hydrophobic, are turned towards the outside of the helix α
 Proteins
Structure and properties
➢ Proteins: Forms

-Collagen

• ¼ of the total protein mass of a mammal is made up of collagen

• Major protein of the extracellular matrix of connective tissue

• Secreted by fibroblasts and by cells of the same origin such as chondroblasts in cartilage and osteoblasts in
bone
 Proteins
Structure and properties
➢ Proteins: Forms

• The primary structure of the molecule has about 1000 residues

• No cysteine => no S-S bridges

• No hydrogen bond

• Stabilized by steric repulsion of proline and hydroxyproline residue

cycles
 Proteins
Structure and properties
➢ Proteins: Forms

-Elastin

• Protein of the extracellular matrix of Connective tissue

• Synthesized by fibroblasts

• Present mainly in tissues whose function requires a certain elasticity (skin, ligaments, arteries, lung tissue)

• Related to collagen by its composition (1/3 of Gly, a lot of proline, no cysteine)

 Proteins
Structure and properties
➢ Proteins: Forms

Globular proteins

• More or less compact spheroid shape

• Alternation of secondary structures organized in space into a tertiary structure

● Structural Classification of Globular Proteins:

• α-domain proteins formed exclusively of α-helices
• β-domain proteins formed exclusively of β-pleated sheets, most often antiparallel (e.g.: Ig)
• Proteins with α and β domains formed by an arrangement of β pleated sheets, most often parallel and α helices
 Proteins
Structure and properties
➢ Protein sequence determination:
How Is the Primary Structure of a Protein Determined?
• The unique characteristic of each protein is the distinctive sequence of amino acid residues in its polypeptide
chain(s).
• It is the amino acid sequence of proteins that is encoded by the nucleotide sequence of DNA.
• Because polypeptide chains are unbranched, a polypeptide chain has only two ends, N-terminal end and C-terminal
end.
• The amino acid sequence is read from the N-terminal end of the polypeptide chain through to the C-terminal end.
• In 1953, Frederick Sanger reported the amino acid sequences of the two polypeptide chains composing the protein
insulin.
 Proteins
Structure and properties
➢ Protein sequence determination:

How Is the Primary Structure of a Protein Determined?

The chemical strategy for determining the amino acid sequence of a protein involves several steps:
1. If the protein contains more than one polypeptide chain, the chains are separated and purified.
2. Intra chain S-S (disulfide) cross-bridges between cysteine residues in the polypeptide chain are cleaved.
3. The N-terminal and C-terminal residues are identified.
4. Each polypeptide chain is cleaved into smaller fragments, and the amino acid composition and sequence of each fragment
are determined.
 Proteins
Structure and properties
➢ Protein sequence determination:
How Is the Primary Structure of a Protein Determined?

Step 1:
• If the protein of interest is a heteromultimer, then the protein must be dissociated into its component polypeptide chains.
• Because subunits in multimeric proteins typically associate through noncovalent interactions, most multimeric proteins
can be dissociated by exposure to pH extremes, or high salt concentrations
• Once dissociated, the individual polypeptides can be isolated from one another on the basis of differences in size and/or
charge.
 Proteins
Structure and properties
➢ Protein sequence determination:

How Is the Primary Structure of a Protein Determined?

Step 2:
• Cleavage of Disulfide Bridges.
• Sulfhydryl compounds such as 2-mercaptoethanol or
dithiothreitol (DTT) reduce S--S bridges to regenerate two
cysteine –SH side chains.
 Proteins
Structure and properties
➢ Protein sequence determination:

How Is the Primary Structure of a Protein Determined?

Step 3:
• Determination of the amino-acid N--Terminus
➢ Method of Sanger - method with DNFB:
• Amino acid (α--amino) + 2.4 DNFB → DNFB-- peptide
• The new bond is resistant to acid hydrolysis
• By acid hydrolysis, all the amino acids are free and the amino acid N--Ter is linked to DNFB (DNP--
N--Ter)
• By chromatography we could identify the amino acid in question
 Proteins
Structure and properties
➢ Protein sequence determination:
How Is the Primary Structure of a Protein Determined?

Step 3:
• Determination of the amino-acid N--Terminus
➢ Method of Sanger - method with DNFB:
 Proteins
Structure and properties
➢ Protein sequence determination:

How Is the Primary Structure of a Protein Determined?

Step 3:

Determination of the N--Terminus amino-acid

➢ Method with Dansyl chloride
• Dansyl chloride reacts with the free amino groups to make a
fluorescent product.
• After acid hydrolysis to produce free amino acids, they are
resolved by chromatography, and detected by fluorescence.
 Proteins
Structure and properties
➢ Protein sequence determination:
How Is the Primary Structure of a Protein Determined?
Step 4:
Determination of the N--Terminus amino-acid
➢ Edman degradation
• Edman reagent: phenylisothiocyanate (PITC)
• PITC reacts with the free amino group, PITC + N- Ter peptide →PITC-peptide
• By action of dilute acid => release only the N--ter (amino acids) which cyclizes.
• Method widely used as the chain remains intact.
• It can recover and be subjected to another cycle with PITC.
• This procedure can be done repetitively, as the covalently modified N--terminal amino acid can be selectively released.
 Proteins
Structure and properties
➢ Protein sequence determination:

How Is the Primary Structure of a Protein Determined?

Step 3:
Determination of the C--Terminus amino-acid
• Enzymatic degradation with carboxypeptidase => It attacks the peptide bond before the amino acid C-Ter
• The C-ter amino-acid is released
• It is isolated and analyzed by liquid chromatography
 Proteins
Structure and properties
➢ Protein sequence determination:

How Is the Primary Structure of a Protein Determined?

Step 4:
• The aim in this step is to produce fragments useful for sequence analysis.
• Specific hydrolysis by proteolytic enzymes
• Enzymatic cleavage of peptides and proteins at defined sites:
• Trypsin is a digestive enzyme that cleaves proteins at peptide bonds that are on the C--side of a Lys or Arg-
• Chymotrypsin is a similar enzyme that cleaves on the C--side of Trp, Tyr, Phe (aromatic residues)
• Trypsin and chymotrypsin are endopeptidases
• Chemical fragmentation
• Br-CN (Cyanogen Bromide) is the most commonly used.
• It attacks Methionine, fragmenting the polypeptide on the C-side of the Met