AMINO ACIDS, PROTEINS

FOR DPT

Dr Umer Saeed
MBBS (King Edward Medical College), M.Phil, PhD biochemistry
Certificate In Medical Teaching (UHS)
 UHS SYLLABUS-DPT

• Amino acids: Classification

• Acid-Base Properties
• Functions & Significance.
• Protein Structure, Primary, Secondary & Super
secondary. &, Structural Motifs
• Tertiary & Quaternary Structures of Proteins
• Protein Domains
• Classification of Proteins
• Fibrous proteins (collagens and elastins ) &
Globular proteins
ACID BASE PROPERTIES
OF AMINO ACIDS
Alanine can exist in 3 forms, at different pH

• Both the α carboxylic & amino groups are ionizable

(can donate or accept proton/H+) at specific pH
• At low pH, both these groups are fully protonated

• Net charge = + ve
 Behavior of ionizable groups in Alanine at
different pH

• As the pH of the solution is raised, the –COOH

dissociates, donating its proton to the medium, thus
forming carboxylate (-COO-) ; net charge = 0
• This is the dipolar form of alanine, and net charge = 0

• This form is also called as ZWITTERION

 What are ‘Ampholytes’?

• The zwitterion can act as either an acid (proton

donor) or a base (proton acceptor), which depending
upon the pH of the medium
• The compounds having both acidic/basic properties
are, therefore, called as Amphoteric electrolytes
(also called as ‘Ampholytes’)
Behavior of ionizable groups in Alanine

• The 2nd ionizable group in alanine is the amino

group
• At higher pH, the amino group also
dissociates, resulting in formation of fully
deprotonated form of alanine
• Net charge = - ve
 Summary:
(ionization of amino acids)

• At acidic pH, the carboxyl & amino groups are

fully protonated, and the amino acid exists as
cation
• Net charge = +ve
 Summary:
(ionization of amino acids)

• At neutral pH, the carboxyl group is de-

protonated, but amino acid is protonated
• Net charge = 0
 Summary:
(ionization of amino acids)

• At alkaline pH, the amino group is also de-

protonated, and the amino acid is in an anion
• Net charge = - ve
 What is ‘ISO-ELECTRIC POINT’?

• Also called as ‘Iso-electric pH (PI)

• DEFINITION: It is that pH at which net charge

on an amino acid is zero (sum of –ve & +ve

charges on an amino acid is zero)

 Alanine has zero net charge at pH=6

• Since Alanine has no net charge at pH=6,

therefore isoelectric point of Alanine is = 6
• At pH = 6, Alanine will have least solubility
 Significance of PI

• If pH of the medium is < PI, then net charge on

an amino acid will be = negative
• If pH of the medium is = PI, then net charge on
an amino acid will be = zero
• If pH of the medium is > PI, then net charge on
an amino acid will be = positive
 Applications of PI

1. Used to separate amino acids from a mixture of

molecules at specific pH in an electric field
2. Used for identification & purification of proteins

3. Used for separation of iso-enzymes

4. Proteins precipitate at PI, as they contain no

charge at that pH
 UHS SEQ (very common)

Q: Define ISOELECTRIC POINT. Give its

significance/applications
 ‘Isoelectric focusing’

• It is a technique, used in biochemistry

laboratory for separating different molecules
based on their PI
 Pka values of amino acids

• Each ionizable group in an amino acid has its

own characteristic pKa value (tendency to lose
protons)
• At this point, the pKa is equal to the pH at
which there is 50% dissociation
Pka values of ionizable groups in an amino
acid

• pK1 = dissociation of –COOH group

• pK2 = dissociation of –NH2 group

• pK3 = dissociation of -R group (Histidine amino acid

has 3 ionizable groups)
 Pka values in glycine

• Glycine is an amino acid with uncharged side

chain and has 2 pKa values

• pK1 due to –COOH

• pK2 due to –NH2

 Pka values in glycine

• Therefore, Glycine can act as a buffer in two

pH ranges
Titration curve of amino acids
Histidine has 3 ionizable groups
Titration curve of histidine
 Amino acids act as buffers

• The –COOH/-COO- pair can serve as a buffer in

the pH region around pK1 and the –NH3/-NH2
pair can buffer in the region around pK2
 MCQS UHS-DPT
• If the pH of the medium is above the
isoelectric point of the amino acid, then the
charge on that amino acid will be:
a. +3
b. +2
c. +1
d. 0
e. -1
 MCQS UHS-DPT
• The amino acid exhibiting three ionizable
groups in its molecule is:
a. Alanine
b. Glycine
c. Histidine
d. Proline
e. Valine
 MCQS UHS-DPT
• In an electric field, if the pH of the medium is
equal to PI, then that amino acid will;
a. Migrate towards anode
b. Migrate towards cathode
c. Remain static
d. Have net positive charge
e. Have net negative charge
 MCQS UHS-DPT
• Which one of the following is regarded as an
imino acid?
a. Proline
b. Valine
c. Histidine
d. Alanine
e. Tyrosine
 MCQS UHS-DPT
• The amino acid which is positively charged at
physiologic pH is:
a. Proline
b. Valine
c. Histidine
d. Alanine
e. Tyrosine
 MCQS UHS-DPT
• The amino acid which is negatively charged at
physiologic pH is:
a. Aspartate
b. Leucine
c. Histidine
d. Valine
e. Alanine
 MCQS UHS-DPT
• The Zwitterions:
a. Act as weak acids
b. Act as bases
c. Act as both acids & bases
d. Are negatively charged molecules
e. Are positively charged molecules
 SEQ UHS-DPT
1. Classify amino acids on the basis of their
structure (-R groups), giving relevant
examples

2. Define isoelectric point

3. Differentiate between essential & non

essential amino acids (or semi essential)
 STRUCTURAL
ORGANIZATION OF
PROTEINS
PRIMARY STRUCTURE OF PROTEIN

• The sequence of amino acids in a protein is

called the primary structure of proteins
• It is important because many genetic diseases
are caused by abnormal amino acid sequences
PRIMARY STRUCTURE OF PROTEIN

• In proteins, amino acids are linked together

covalently by PEPTIDE BONDS
• These are amide linkages between the α-
carboxyl group of one amino acid and the α-
amino group of another
PRIMARY STRUCTURE OF PROTEIN

• The sequence of nucleotides in a protein-

coding region in DNA specifies the amino acid
sequence of a peptide
 SECONDARY STRUCTURE OF PROTEIN

• The arrangements of amino acids in a protein

is called the secondary structure of proteins
• The polypeptides contain regular
arrangements of amino acids that are located
near each other
 SECONDARY STRUCTURE OF PROTEIN

• These arrangements are:

• α helix
• β sheets
• β bends (or β turns)
 α- HELIX

• It is the most common arrangement

• It is rigid, right handed, spiral structure,
consisting of tightly packed, coiled
polypeptide backbone core, with the side
chains extending outwards
 Hydrogen bonds in α helix

• Helical structure is mainly stabilized by

hydrogen bonds between the peptide bond
carboxyl oxygens and amide hydrogens that
are part of the polypeptide backbone
 3.6 amino acids per turn of helix

• Each turn of α helix, contains 3.6 amino acids

 Amino acids that disrupt the α helix

• Proline amino acid inserts a kink in the chain,

interfering with the smooth, helical structure
 Amino acids that disrupt the α helix

• Glycine amino acid also breaks helix because

of its R-group
 Amino acids that disrupt the α helix

• Valine amino acid has low propensity for α

helix
 β- SHEET

• It is an arrangement in which all the peptide

bond components are involved in hydrogen
bonding
• Because the surfaces of β sheets appear
‘pleated’ they are often called as ‘pleated
sheets’
 Formation of β- sheet
• A β sheet is formed by two or more peptide
chains (β strands), aligned laterally and
stabilized by hydrogen bonds between the
carboxyl & amino groups of amino acids that are
either far apart in a single polypeptide (intra-
chain bonds) or are in different polypeptide
chains (inter-chain bonds)
 3. β- BENDS

• β bends reverse the direction of a polypeptide

helping it to form a globular shape
• These are usually formed by 4 amino acids,
one of which is mostly proline (or glycine)
 SUPER SECONDARY STRUCTURES
‘motifs’

• Globular proteins are formed by combination

of secondary structural elements (α helix, β
sheets & coils) producing specific geometric
patterns called as MOTIFS
 TERTIARY STRUCTURE OF PROTEINS
(folding of proteins)

• Tertiary structure refers to folding of domains

(subunits) and final arrangement of these
domains in the polypeptide
 TERTIARY STRUCTURE OF PROTEINS
(folding of proteins)

• Tertiary structure is determined by the

primary structure of a polypeptide chain
DOMAINS:
• Domains are the functional & three
dimensional structural units of polypeptides
 FORCES WHICH STABILIZE TERTIARY
STRUCTURE

• The three dimensional structure of each

polypeptide is determined by its amino acid
sequence
• Interactions among the amino acid side chains
guide the folding of the polypeptides to form a
compact structure
 FORCES WHICH STABILIZE TERTIARY
STRUCTURE

1. Disulfide bonds:
• These covalent linkages are formed (-S-S-)
form the sulfhydryl groups between 2
cysteine amino acids
 FORCES WHICH STABILIZE TERTIARY
STRUCTURE

2. Hydrophobic interactions:
• Amino acids with non-polar side chains tend
to be located in the interior of the
polypeptide molecule, where they associate
with other hydrophobic amino acids
 FORCES WHICH STABILIZE TERTIARY
STRUCTURE

3. Hydrogen bonds:
• Amino acids side chains containing oxygen or
nitrogen-bound hydrogen which form
hydrogen bonds
 FORCES WHICH STABILIZE TERTIARY
STRUCTURE

4. Ionic interactions:
• Negatively charged amino acids (glutamate,
aspartate) can interact with positively
charged groups (lysine, histidine, arginine)
 PROTEIN FOLDING

• Interactions between the side chains of amino

acids determine how a linear polypeptide
chain folds into the three dimensional shape
of the functional protein
 Protein denaturation

• Denaturation results in unfolding of a

polypeptide without the hydrolysis of peptide
bonds
• Agents which cause denaturation: heat, urea,
organic solvents, strong acid/bases,
detergents
 What are ‘chaperones’?

• These are specialized group of proteins (also

called as ‘Heat shock proteins), which interact
with polypeptide at various stages during the
folding process
 QUATERNARY STRUCTURE OF PROTEINS

• Arrangement of polypeptide chains subunits is

called as Quaternary structure of proteins
• Many proteins consist of a single chain and are
called monomeric proteins
 QUATERNARY STRUCTURE OF PROTEINS

• Some proteins contain two or more

polypeptide chains
• The subunits are held together mainly by non-
covalent interactions such as hydrogen bonds,
ionic bonds, hydorphobic interactions
 Protein misfolding

• Protein folding is a complex process that can

sometimes result in improperly folded
molecules
• Usually these unfolded proteins are tagged
and degraded by cellular mechanisms
 Protein misfolding

• However, if these improperly folded proteins

are not removed adequately, they tend to
accumulate & form aggregates
• Deposits of these aggregated proteins cause
some important diseases
Misfolded proteins form aggregates
Protein misfolding
diseases
 Misfolded protein aggregates
AMYLOID

• Misfolding of proteins may be spontaneous or

caused by mutations which produce altered
proteins
• The aggregates of these misfolded proteins
are termed as AMYLOIDS
 ALZHEIMER DISEASE
A protein misfolding disease
• In AD, there is deposition of amyloid in certain
parts of the brain causing neruo-degenerative
features
 ALZHEIMER DISEASE
A protein misfolding disease

• The dominant component of the amyloid

aggregate is the amyloid β (Aβ) which is an
extracellular peptide containing 40-42 amino
acid residues
Aβ is neurotoxic
 ALZHEIMER DISEASE
A protein misfolding disease

• Aβ is derived by enzymatic cleavage of a large

amyloid precursor protein, which is expressed
on the surface in brain
• The Aβ peptides aggregate, generating the
amyloid found in the brain tissue
 ALZHEIMER DISEASE
A protein misfolding disease

• Another factor involved in the development of

AD is the accumulation of ‘neurofibrillary
tangles’ inside the neurons
• Key component of these tangles is the
abnormal form of ‘Tau’ protein
clinical features of AD
 PRION DISEASES
(protein misfolding disease)

• The PRION PROTEIN causes diseases such as:

• Mad cow disease in cattle
• Jakob disease in humans
• Scrapie in sheep
 PRION DISEASES
(protein misfolding disease)

• Changes in the three dimensional

conformation in prion proteins makes them
‘infectious’ such that the some α helices are
replaced by β sheets, making them highly
resistant to proteolytic degradation
 MCQS UHS-DPT
• The two amino acids most frequently present
at beta bends are:
a. Proline & lysine
b. Valine & glycine
c. Tryptophan & alanine
d. Histidine & valine
e. Proline & glycine
 MCQS UHS-DPT
• The amino acids present at each turn of an α
helix are:
a. 1.4
b. 2.6
c. 2.8
d. 3.4
e. 3.6
 MCQS UHS-DPT
• In Sickle cell disease, primary defect is in:
a. Primary structure
b. Secondary structure
c. Super secondary structure
d. Tertiary structure
e. Quaternary structure
 MCQS UHS-DPT
• The bonds which primarily stabilize the
primary structure of proteins are:
a. Hydrogen bonds
b. Covalent bonds
c. Hydrophobic bonds
d. Ionic bonds
e. Non-covalent bonds
 MCQS UHS-DPT
• Which of the following stabilizes the α helix
structure of proteins?
a. Hydrogen bonds
b. Peptide bonds
c. Vander waals forces
d. Hydrophobic interactions
e. Covalent linkages
 MCQS UHS-DPT
• The specific proteins which assist in proper
folding of a native protein are:
a. Peptones
b. Prolamines
c. Chaperons
d. Phosphoproteins
e. Ceruloplasmin
 MCQS UHS-DPT
• The amino acid which imparts positive charge
to a protein at physiologic pH is:
a. Arginine
b. Alanine
c. Proline
d. Tryptophan
e. Valine
 MCQS UHS-DPT
• Primary pathology in Alzheimer disease is:
a. Deletion of amino acid
b. Substitution of amino acid
c. Deposition of misfolded protein
d. Formation of prion protein
e. Denaturation of proteins in brain
 MCQS UHS-DPT
• Many proteins consist of a single chain, and
are called monomeric proteins. This level of
structure is studied in:
a. Primary structure
b. Secondary structure
c. Super secondary structure
d. Tertiary structure
e. Quaternary structure
 SEQS UHS-DPT

1. Briefly give structural features of different

types of repetitive secondary structures of
proteins
2. Differentiate clearly between standard and
non-standard amino acids
 SEQS UHS-DPT

3. Differentiate clearly between complete & incomplete

proteins

4. Differentiate clearly between denaturation &

coagulation

5. How peptide bonds are formed? Give their properties,

and name any one agent which breaks these bonds
CLASSIFICATION OF
PROTEINS
BASED ON PHYSICOCHEMICAL PROPERTIES

1. Simple proteins

2. Conjugated proteins

3. Derived proteins
 Simple proteins

• These are the proteins which on hydrolysis

yield only amino acids
• These include:

• Albumins, Globulins, Protamines, Histones,

Globins, Gliadins or prolamines,
Albuminoids/scleroproteins
 Albumin

• Albumin is a protein with high biological value

• Soluble in water & is heat coagulable

• Examples include;

• Egg albumin, lactalbumin, serum albumin

 Globulins

• These are also of high biological value and are

heat coagulable
• Examples include;
• Ovoglobulin, lactglobulin, serum globulin,
legumin in legumes
 Protamines

• These are soluble in water, but not coagulable

by heat
• Protamines are highly basic protein (rich in
arginine)
• Examples include;

• Present in sperm cells of salmon and sardine

 Histones

• Histone proteins are soluble in water, and are

not coagulable by heat
• These are also basic proteins (rich in arginine)

• Examples include;

• In combination with nucleic acids to form

nucleoproteins
 Globins

• Globin proteins are rich in histidine

• It is present in combination with heme to form

hemoglobin (myoglobin)
 GLIADINS OR PROLAMINS

• Soluble in ethanol but are insoluble in water

• Examples include;
• Gliadin present in wheat and zein of maize
 ALBUMINOIDS OR SCLEROPROTEINS

• Insoluble in water and dilute acids

• These are exclusively animal proteins and form

supporting structures and connective tissues
• Examples include;

• Keratin, collagen and elastin

 Conjugated proteins
• These are composed of proteins & a non-
protein part (prosthetic part)
1. Phosphoproteins
• These are proteins attached with phosphoate
ions
• These include casein of milk, vitellin of egg yolk
 Conjugated proteins
2. Lipoproteins
• These are proteins attached with lipids

• Lipoproteins transport various lipids in blood

circulation
• For example; chylomicrons, VLDL, LDL, HDL
 Conjugated proteins
3. Nucleoproteins
• These are proteins (histones) attached with
nucleic acids (DNA, RNA)
• These include; histones
 Conjugated proteins
4. Metalloproteins
• These are proteins attached with metallic
ions
• For example; Hemoglobin, myoglobin (Fe+2),
Ceruloplasmin (Cu+2), Carbonic anhydrase
(Zn+)
 Conjugated proteins
5. Chromoproteins
• These are proteins attached with colored
pigments
• For example; Hemoglobin, myoglobin,
Rhodopsin (visual pigment), melanin (skin)
 Conjugated proteins
6. Glycoproteins
• These are proteins attached with
oligosaccharides
• These include; Immunoglobulins, hormones
(LH, FSH, TSH), plasma proteins (transferrin,
ceruloplasmin)
 Derived proteins

• These are produced from simple and

conjugated proteins
• Further subdivided into:

1. Primary derived proteins

2. Secondary derived proteins

 Primary derived proteins

• These are denatured or coagulated proteins

• Denaturation is the loss of the three dimensional

structure which causes loss of function of the protein
• Denaturation occurs when protein loses its native
secondary, tertiary, quaternary structure, but the
peptide bonds are not broken
 Agents causing protein denaturation

• Heat

• Vigorous shaking

• Extreme pH/temperature

• Salts of heavy metals

• Urea

• Organic solvents such as alcohol

 Secondary derived proteins

• These are formed as intermediates during the

hydrolysis of proteins
• These are divided into:
a. Proteoses
b. Peptones
c. Peptides
 CLASSIFICATION BASED ON MOLECULAR
LENGTH/SHAPE

• Proteins are classified according to their

overall shape and length

• Based on AXIAL RATIO → length/breadth

 CLASSIFICATION BASED ON MOLECULAR
LENGTH/SHAPE

• According to axial ration proteins are classified

as:
• Fibrous proteins (axial ratio > 10)
• Globular proteins (axial ratio < 10)
 CLASSIFICATION BASED ON MOLECULAR
LENGTH/SHAPE

• Examples of fibrous proteins: collagen, elastin

• Examples of globular proteins: hemoglobin,

enzymes, albumin
 CLASSIFICATION BASED ON FUNCTIONS

1. Regulatory proteins: these regulate

biochemical reactions, for example; insulin,
epinephrine

2. Contractile proteins: these include skeletal

muscle proteins for muscle contraction, for
example; actin, myosin
 CLASSIFICATION BASED ON FUNCTIONS

3. Transport proteins: these transport

substances in circulation for example;
hemoglobin, albumin

4. Catalytic proteins: these catalyze reactions,

and include enzymes, for example; amylase,
lipase etc
 CLASSIFICATION BASED ON FUNCTIONS

5. Immune proteins: these include

immunoglobulins (IgG, IgA, IgM, IgE)

6. Structural proteins: these are structural

components of our tissues/organs ,for
example; collagen, elastin
 CLASSIFICATION BASED ON FUNCTIONS

7. Storage proteins: these proteins have storage

functions, for example ferritin (iron),
Ceruloplasmin (copper)

8. Genetic proteins: these are present in

combination with DNA, RNA, for example
histone proteins
CLASSIFICATION BASED ON NUTRITIONAL
VALUE

1. COMPLETE PROTEINS (high value proteins):

these contain all the essential amino acids
required for synthesis of tissue proteins in
human body
• For example; animal proteins/eggs, meat etc
(except gelatin)
CLASSIFICATION BASED ON NUTRITIONAL
VALUE

2. INCOMPLETE PROTEINS (low value proteins):

these are deficient in one or more essential
amino acids
• For example; vegetable proteins (peas,
legumes, beans etc)
Vegetable proteins are deficient in one or more
essential amino acid

• Wheat → Lysine

• Corn → Lysine, tryptophan

• Beans/peas → Methionine

• Rice → Lysine
 MCQS UHS-DPT
• Hemoglobin is an example of:
a. Transport protein
b. Regulatory protein
c. Structural protein
d. Immune protein
e. Storage protein
 MCQS UHS-DPT
• An example of regulatory protein among the
following is:
a. Ferritin
b. Vitellin
c. Casein
d. Pepsin
e. Insulin
 MCQS UHS-DPT
• Nucleosomes are formed by binding of DNA
with proteins:
a. Transferrin
b. Albumin
c. Histones
d. Casein
e. Prolamines
 MCQS UHS-DPT
• A person is using corn as a staple diet, the
protein which might be deficient in this person
is:
a. Alanine
b. Proline
c. Arginine
d. Tryptophan
e. Tyrosine
 MCQS UHS-DPT
• Collagen is the most abundant protein in
human body, and this protein is an example
of:
a. Storage protein
b. Transport protein
c. Structural protein
d. Regulatory protein
e. Catalytic protein
 MCQS UHS-DPT
• Fibrous proteins are those having an axial ratio
greater than:
a. 2
b. 4
c. 6
d. 8
e. 10
 MCQS UHS-DPT
• Identify a fibrous protein among the following:
a. Hemoglobin
b. Elastin
c. Albumin
d. Ceruloplasmin
e. Ferritin
 MCQS UHS-DPT
• In Alzheimer disease, there is deposition of
abnormal proteins which are neurotoxic, and
possess primarily in their structure:
a. β sheets
b. α helix
c. Both α helix and β sheets
d. β bends and turns
e. Tripple helix
 MCQS UHS-DPT
• The protein which has least nutritional value
is:
a. Egg white
b. Milk
c. Chicken
d. Gelatin
e. Fish
 MCQS UHS-DPT
• The phosphoprotein among the following is:
a. Collagen
b. Albumin
c. Ferritin
d. Casein
e. Ceruloplasmin
 MCQS UHS-DPT
• Wheat is deficient in amino acid:
a. Lysine
b. Methionine
c. Tryptophan
d. Proline
e. Arginine
 MCQS UHS-DPT
• In pathological prion proteins, the underlying
defect in protein structure is:
a. Disruption of α helices
b. Replacement of some α helices with β
sheets
c. Loss of some β sheets
d. Loss of some α helices
e. Degradation of many hydrogen bonds
 SEQS UHS-DPT

1. Give a brief account of structural features of

α helix & β sheets

2. Write three important functions of albumin.

Give any two causes of hypoalbuminemia

3. Classify proteins on the basis of functions

 SEQS UHS-DPT

4. What is meant by tertiary structure of

proteins? Enlist the forces which stabilize
this structure

5. What are ‘Chaperones’? Give their

biochemical significance