proteins: structure ,types and purification techniques

1. Proteins : structure ,types
and purification techniques

2. What is Protein??
 Protein is a macro nutrient composed of
monomer form amino acids that is
necessary for the proper growth and
function of the human body.
 Amino acids are small molecules that
contain carbon, hydrogen, oxygen, and
nitrogen atoms; two also contain sulfur
atoms.

3. Each amino acid has 4 different groups
attatched to α- carbon ( which is Catom
next to COOH).These 4 groups are :
amino group, COOH group , Hydrogen
atom and side chain(R).

4. Stereochemistry of
amino acids
•Configuration
•For all the common amino acids
except Glycine, the α-carbon is
bonded to four different groups.
• The α-carbon atom is thus a chiral
center.


5. The two enantiomers of each amino acid are designated
by D, L system according to the D- and L-glyceraldehyde.
 D: Dextrorotation;
 L:Levorotation
•Only the L-amino acids have been found in proteins.
•(D-isomers have been found only in small peptides of bacteria cell
walls or in some peptide antibiotics).

6. Classification of Amino
Acids
Based on polarity
Nonpolar, aliphatic
amino acids
Polar, uncharged amino
acids
Aromatic amino acids
Acidic amino acids
Basic amino acids

7. Nonpolar, aliphatic
amino acids
•Gly, Ala, Pro, Val, Leu, and
Ile

8. • Gly:
• R group: hydrogen.
• symmetric, not
chiral.
• Pro:
Imino acid.
five-membered ring
structure, rigid in
conformation.

9.  Ala, Val, Leu, and Ile
Hydrocarbon R groups, often involved in
hydrophobic interactions for stabilizing
protein structure .

10. Polar, uncharged
amino acids
 Ser, Thr, Cys, Asn, Gln and Met.
• R
gro
up:
Hydr
ophili
c
•H
y
dr
o
x

11. Aromatic amino acids
Phe, Tyr and Trp.
Phe and Tyr: benzene rings.
Tryptophan: indole ring.
•The -OH group in Tyr is an important functional group
in proteins. (phosphorylation, hydrogen bond, etc),
polar

12. •They are jointly responsible for the light
absorption of proteins at 280 nm.
•Proteins in solution
absorb UV light with
absorbance
maximum at 280nm.
•Measuring
protein content
by photo
spectrometry.

13. Acidic amino acids
Asp and Glu
Have carboxyl in their R groups.

14. Basic amino acids
Lys, Arg, and His.
R groups
Amino
Guanidino
Imidazole
Positive
charged R
groups at pH 7.0

15. Essential and non-essential amino acids
 Essential amino acids (or indispensable amino
acids):
• Cannot be synthesized by the humans, must
be supplied in the diet
• 8: Phe, Val, Thr, Trp, Ile, Met, Leu, Lys
 Semi-essential amino acids:
• 2: His and Arg
• Required by infants and growing children

16. Acid/base properties of AAs
 Amino acid has both a basic amine group
and an acidic carboxylic acid group.
 In neutral solution (pH 7.0), the amino
acid contains a negative charge and a
positive charge. It is called a zwitterion
(German for “hybrid ion”).

17. •AAs ionize to various states depending on
pH values.
• pI: there is a specific pH (designated
isoelectric point, pI)
at which an AA has equal positive and
negative charge (no net electric charge) .
cati zwitter ani
+
H+
+
H+
R—CH—
COOH
|NH
+
R—CH—
COO-
|NH
+
R—CH—
COO-
|
NH
-
H+
-
H+
A
+
A
0
A
-
3
pH<
pI
3
pH=
pI
2
pH>
pI

18. peptide
bond
Amino
acid
resid
ue
Polypeptides
Peptide bond: the special name given to the amide bond between
the carboxyl group of one amino acid and the -amino group of
another.

20. The peptide chain is directional.
 Amino-terminal or N-terminal: the end having a free a-amino
group.
 Carboxyl-terminal or C-terminal: the end having a free a-
carboxyl group.
 By convention, the N-terminal is taken as the beginning of
the peptide chain,
• and put at the left (C-terminal at the right).

21. Polymers of amino acids
 Peptides can be classified according to
how many amino acids they contain
 Dipeptide: 2 amino acid residues,
 Tripeptide: 3 residues, and so on
 Oligopeptide: 12~20 residues
 Polypeptide: many residues

22. Proteins have different levels of
organisation
 Primary Structure
Secondary Structure
Tertiary Structure
Quaternary Structure

23. Primary
Secondary
Tertiary
Quaternary
Assembly
Folding
Packing
Interaction
Protein Structure

24.  The primary structure of protein refers to
the sequence of amino acids present in
the polypeptide chain.
 Amino acids are covalently linked
by peptide bonds or covalent
bonds.
 Each component amino acid in a
polypeptide is called a"residue”
or “moiety”.
 By convention the primary structure of
protein starts from the amino terminal
(N) end and ends in the carboxyl
Primary Structure

25. It is a local, regularly
occurring structure in
proteins and is mainly
formed through hydrogen
bonds between backbone
atoms.
Pauling &Corey studied
the secondary structures
and proposed 2
conformations
o αhelix
o βsheets.
Secondary Structure

26.  Right handed spiral
structure.
 Side chain extend outwards.
 Stabilized by H bonding that
are arranged such that the
peptide Carbonyl oxygen
(nth residue) and amide
hydrogen(n+4 th residue).
 Amino acids per turn – 3.6
 Alpha helical segments,
are found in many
globular proteins like
myoglobin,troponin C.

29. o Formed when 2 ormore
polypeptides line up sideby
side.
o Individual polypeptide –
beta strand.
o Each beta strand is fully
extended.
o They are stabilized by
hydrogen bond between N-
H and carbonyl groups of
adjacent chains.

30. o Beta sheets come in two varieties
Antiparallel beta sheet – neighboring hydrogenbonded
polypeptide chains run inopposite direction.
Parallel beta sheet- hydrogen bonded chains extend in the same direction.
Theconnection between two antiparallel strands may be just a small
loop but the link between tandem parallel strands must be a
crossover connection that is outof the plane of the βsheet.

32.  The tertiary structure defines the specific overall 3-D shape
of the protein.
 Tertiary structure is based on various types of interactions between
the side-chains of the peptide chain

33. In globular proteins
Tertiary interactions are frequently stabilized by
sequestration of hydrophobic amino acid
residues in the protein core.
Consequent enrichment of charged or
hydrophilic residues on
the protein's water-exposed surface.
In secreted proteins
disulfide bonds between cysteine residue helps to
maintain the protein's tertiary structure

34. Interactions stabilizing tertiary
structure :
1. Disulfide bonds
1. Hydrophobic interactions
2. Hydrogen bonds
3. Ionic interactions

35. Covalent bond
between sulfur
atoms on two
cysteine amino
acids.

36.  H bonds are weak which
allows to be broken and
reformed easily.
 Allows structural change
and produces
‘functional’molecules

38.  Close attraction of
non-polar Rgroups
through dispersion
forces.
 They are non
attractive
interactions, but
results from the
inability of water to
form hydrogen
bonds with certain
side chains.
 Very weak but
collective

40.  The quaternary protein structure involves
the clustering of several individual peptide
or protein chains into a final specificshape.
 Avariety of bonding interactions including
hydrogen bonding, salt bridges, and disulfide
bonds hold the various chains into a particular
geometry.
 Two kinds of quaternary structures: both aremulti-
subunit proteins.
Homodimer :association between identical
polypeptide chains.
Heterodimer:interactions between subunits of very
different structures.
 The interactions within multi subunits are the same
as that found in tertiary andsecondary structures

41. o Haemoglobin is a globular
protein with 4 polypeptide
chains bonded
together. It therefore
has a quartenary
structure.
o There are 4haem groups
each contain iron.
o Each haem group can carry
one molecule of oxygen.
o The four polypeptide chain
consistsof
two alpha and two betachains.

43. The basic aim in protein purification is to
isolate one particular protein of interest from
other contaminating proteins to study its
structure and function, increasing its stability
and large scale production
Protein Purification
Techniques

44. Protein Purification
• Purification of proteins is an essential first step
in understanding their function
• Proteins must be released from the cell to be
purified
• Based on the basic properties of proteins like
solubility, size, charge and binding affinity

45. General Steps in Protein Purification
Selection of a
protein source
Assay
of
Proteins
Homogenization
and
Solubilization
Stabilization
of Proteins
Detergent e.g.
Triton X
100
pH 7 and
temperature
below 25°C
Radioimmunoassa
y, ELISA, Western
Blotting

46. 1. Ammonium Sulfate Precipitation
• This technique exploits the fact that the
solubility of most proteins is lowered at high
salt concentrations
• The concentration of ammonium sulfate at
which a particular protein comes out of
solution and precipitates is different from
other proteins in the mixture

47.  Ammonium sulfate is an inorganic salt with a high solubility
 that disassociates into ammonium (NH4+) and sulfate (SO42−) in
aqueous solutions.
 Ammonium sulfate is especially useful as a precipitant
because it is highly soluble, stabilizes protein structure, has a
relatively low density, is
readily available, and is relatively inexpensive.

48. Salting IN, Salting OUT
Protein solubility is
affected by ions. At low
ion concentrations
(<0.15 M), protein
solubility increases along
with ionic strength. Ions in
the solution shield protein
molecules from the charge
of other protein molecules
in what is known as
‘salting-in’. At a very high
ionic strength, protein
solubility decreases as
ionic strength increases in
the process known as
‘salting-out’. Thus, salting
out can be used to separate

49. 2. Dialysis
• A semipermeable membrane is used to remove
small molecules such as salts and ammonium
sulfate from a protein solution
• Based upon size of molecules

51. 3. Gel Filtration Chromatography
• separate proteins according to their size. Also
termed as “sizeexclusionchromatography”
• A gel filtration column has beads with
channels running through them e.g. agarose

53. • Smaller molecules
can freely enter the
internal solvent
space of the gel
bead
• Larger molecules are
too large to
penetrate the gel
pores and travel
between beads and
elute first

56. 4. Ion exchange chromatography
• separate proteins on basis of their over all
(net) charge
• Retention is based on electrostatic interaction
between solute ions and fixed ion charge on
the stationary phase
Cation
Exchange
Chromatograph
y
Anion
Exchange
Chromatograph

57. Anion exchange resins
(positive charge) separate
negatively charged
compounds
Cation exchange resins
(positive charge) separate
positively charged
compounds

60. Affinity
chromatography
Affinity chromatography is a method of separating biochemical
mixture based on a highly specific interaction between antigen
and antibody, enzyme and substrate, receptor and ligand, or
protein and nucleic acid
• Based upon molecular conformation
• Exploits the specific, high affinity
• Ligands function in a fashion similar to that of antigen-
antibody interactions

62. Affinity His-Tag Purification

63. Affinity GST-Tag Purification

65. The ligand is immobilized onto a
solid support matrix
The crude extract is passed
through the column.
The target molecule for which
the ligand possesses affinity is
retained
All other material is eluted
The bound target protein is
eluted by alteration of the
mobile-phase conditions.

66. Proteins change their shape when exposed to different pH or
temperatures. The body strictly regulates pH and temperature to
prevent proteins such as enzymes from denaturing. Some
proteins can refold after denaturation while others cannot.
Chaperone proteins help some proteins fold into the correct
shape.
Protein Denaturation

68. Module II : Carbohydrates: Structure, Function

69. Definition
 Carbohydrates may be
defined as polyhydroxy
aldehydes or ketones or
compounds which produce
them on hydrolysis.
 Formula = (C.H2O)n

70.  Carbohydrates are organic molecules
found innature, constituting one of the
four major classes of biomolecules.
 The other three are proteins, nucleic acids
andlipids.‐
 Saccharides (saccharo is Greek for
―sugar)

71. Straight
chain
Ring
structure
Chair
form

72. Classificati
on
1
Monosaccharide
2
Oligosaccharide
3
Polysaccharide

73. Monosaccharide
Cannot further Hydrolyzed

75. Sucrose
 Lactose
 Maltose
Isomaltose
Disaccharides

76. Sucro
se
It is thesweeteningagentknown ascane
sugar.It is presentinsugarcaneand
various fruits.

77. Lactose
It is thesugar presentin
milk

78. Malto
se

79. Glycosidic linkage
Glycosyltransferases

80. Oligosaccharide
• Oligosaccharides(Greek: oligo-few) contain 2-
2O
monosaccharide molecules
• Joined by glycosidic bond

82. Polysaccharides
Contain more than 20 monosaccharides

83. Polysacchari
des

85. Polysacchari
des
Homopolysaccharides
Starch
Glycogen
Cellulose
Inulin
Dextrans
Heteropolysacchr
ides
Heteropolysaccharides
Agar
Mucopolysacc
haride

86. Homopolysaccharides
Starch
Glycogen
Cellulose
Inulin
Dextrans
Chitin

87. Starc
h
 Starches are carbohydrates in which 300 to
1000 glucose units join together. It is a
polysaccharide which plants use to store
energy for later use.
 It is the reserve carbohydrate of plant
kingdom.
 Sources: Potatoes, cereals (rice, wheat)
and other food grains.
 Starch is composed of amylose and

90. Glycogen
 The most common homopolymer in animal cells is
glycogen, the storage form of glucose.
 It is thereserve carbohydrate inanimals.It is stored inliver
andmuscle.
 About 5% ofweightof liveris madeupbyglycogen.
 Excess carbohydratesaredeposited as glycogen.

91. Cellulo
se
 Itismadeupofglucoseunits combinedwithbeta-1,4 linkages.
It hasastraightlinestructure,withno branchingpoints.
 The multiple hydroxyl groups on the glucose from one
chain form hydrogen bonds with oxygen atoms on the
same or on a neighbor chain, holding the chains firmly
together side-by-side and forming microfibrils with
high tensile strength. This confers tensile strength in
cell walls where cellulose microfibrils are meshed into
a polysaccharide matrix.
 Beta-1,4bridgesarehydrolyzedby theenzymecellobiase. But

94. Dextra
ns
 These arehighlybranchedhomopolymers ofglucoseunitswith
α(1→2),α(1→3),α(1→4)andα(1→6)-linkage.
 Theyareproduced bymicro- organisms.
 Dextraniscommerciallyavailableanditisusedasdrugs,
especiallyasbloodplasmavolumeexpander.
 Ithasfoundindustrialapplicationinfood,pharmaceuticaland
chemicalindustriesasadjuvant,emulsifier,carrierandstabilizer.
Infoodindustrydextraniscurrentlyusedasthickenerforjam
andice-cream.Itpreventscrystallizationofsugar,improves
moistureretentionandmaintainsflavorandappearanceof
variousfooditems.

96. Glycoprotei
ns
• Proteoglycans:When carbohydrate
• chainsare attachedto apolypeptide
• chain.
• Glycoproteins:Carbohydrate
• content≤ 10%.

97. What is Glycoprotein ?:
 Glycoproteins are proteins that contain
oligosaccharide chains (glycans)
covalently attached to polypeptide side-
chains.
This process is known as glycosylation.
The carbohydrate is attached to the protein
during Post-translational modification.
 In proteins that have segments extending
extracellularly, the extracellular segments are
often glycosylated.
Glycoproteins

99. Glycoproteins and Proteoglycans
Glycoproteins
Proteins conjugated to
saccharides lacking a Protein >> carbohydrate
serial repeat unit
Proteoglycans
Proteins conjugated to
polysaccharides with
serial repeat units
Carbohydrate >> protein

100. 2 major classes:-
1) N-linkage (N -acetylglucosamine to asparagine)
2) O-linkage (N -acetylgalactosamine to serine)
 N-glycosidic linkage(ie, N-linked), involving the amide nitrogen
of asparagine and N -acetylglucosamine (GlcNAc-Asn)
 O-glycosidic linkage(ie, O-linked), involving the hydroxyl
side chain of serine or threonine and a sugar such as N -
acetylgalactosamine (GalNAc-Ser/Thr)
CLASSIFICATION OF
GLYCOPROTEINS:-

102. The sequence is an Asn-X-Ser or Asn-X-Thr sequence, where X is any amino acid
except proline and the glycan may be composed of N-acetylgalactosamine,
galactose, N-acetylglucosamine, mannose, and other monosaccharides.

103.  O-linked glycosylation is the attachment of a sugar molecule to
the oxygen atom of serine (Ser) or threonine (Thr) residues in a
protein.
 O-glycosylation is a post-translational modification that occurs
after the protein has been synthesised.
 In eukaryotes it occurs in the Golgi apparatus and occasionally
in the cytoplasm; in prokaryotes, it occurs in the cytoplasm.

106. Glycopeptide bonds
Type I
Type II Type III
N-Glycosyl linkage to Asn
O-Glycosyl linkage to Ser (Thr) O-Glycosyl linkage to 5-HOLys
O
H
OH H
H
CH2OH
H
OH
HN C CH2 CH COOH
O
H HN C CH3
NH2
O
O
H
OH
O
H
H
HNH
CH2OH
H
OH
CH2
NH2
CH COOH
Ser
C CH3
O
O
H
OH
O
H
H
OHH
CH2OH
H
OH
CH
CH2
CH2
CH2
NH2
CH COOHH2N
Glc
NAc
Asn
Glc
NAc
HOLysGlc

107. Functions Served by
Glycoproteins
Function Glycoproteins
Structural molecule Collagens
Lubricant and protective agent Mucins
Transport molecule Transferrin, ceruloplasmin
Immunologic molecule Immunoglobulins, histocompatibility
antigens
Hormone Chorionic gonadotropin,
thyroid- stimulating hormone
(TSH)
Enzyme Various, eg, alkaline phosphatase
Cell attachment-recognition site Various proteins involved in cell-cell
(eg, sperm-oocyte), virus-cell,
bacterium-cell, and hormone-cell
interactions

108. Antifreeze Certain plasma proteins of cold-water fish
Interact with specific carbohydrates Lectins, selectins (cell adhesion lectins),
antibodies
Receptor Various proteins involved in hormone and
drug action
Affect folding of certain proteins Calnexin, calreticulin
Regulation of development Notch and its analogs, key proteins in
development
Hemostasis (and thrombosis) Specific glycoproteins on the surface
membranes of platelets

109. Glycosaminoglycans / GAGS
10
9
o r Mucopolysaccharides
 Are large complex of –ve charged (carboxy & sulfate
groups) heteropolysaccharide chain generally
associated with a small amount of protein -
proteoglycan.
 Special ability to bind large amount of water producing
gel like matrix, that forms the bodies groundsubstance.
 Unbranched, long repeating diasaccharide Contains
uronic acid & amino sugars.

110.  Amino sugar – Glucosamine or Galactosamine.
11
0
Uronic acid – Glucuronic acid.

111. Classificati
on
GAGS
Sulphate free Sulphate containing
Hyaluronic acid
11
1
Chondrotin
Sulphate keratan
sulphate Heparin
Heparan Sulphate

112. • Contains D-Glucoronic acid +
Galactosamine.
• Most abundant GAG in body.
1. Chondroitin s u l f a t e
11
2

113.  Widely distributed in bone, cartilage &tendons.
Function :
 In cartilage, it binds collagen & hold fibers in a tight
strong network.
 Role in Compressibility of cartilage in weight bearing
along with Hyaluronicacid.
2types of chondroitin sulfate:
 Sulphated at C 4 or C 6 group.
11
3

114. 2. Hyaluronic acid
Contains D-Glucoronic acid +Glucosamine.
It is sulphate free GAG.
It is sulphate free GAG.
11
4

115.  Ground substance of synovial fluid of joints, vitreous
humor of eyes and connective tissues,tendon.
 Hyaluronidase is an enzyme that breaks β-1 –4 linkages.
 Present in high concentration in testes, seminal fluid, & in
certain snake and insect venoms.
11
5

116. Functions of Hyaluronic acid
11
6
 Serves as a lubricant and shock absorbant in joints.
 Determines charge selectiveness of renal glomerulus.
 Acts as seives in extracelluarmatrix.
 Permits cell migration during morphogenesis & wound repair.
 Hyaluronidase enzyme of semen degrades the gel aroundovum
& allows effective penetration of sperm into ovum.

117. 3. Heparin
• Contains D-Glucuronic acid +
Glucosamine
• it is the only intracellular GAG.
11
7

118. It is an anticoagulant (prevents blood clotting )
Found in granules of mast cells that line the arteries of
lung, liver, kidney, spleen.
Strongly acidic due to presence of more sulphategroup.
Heparin helps in the release of the enzyme lipoproteinlipase.
Helps to clear the lipidemia after fatty meal – also calledclearing
factor.
11
8

119. 4. Keratan Sulfate
•Contain D-Glucose + Glucosamine
Only GAG with no uronic acid.
11
9

120.  Found in cornea & tendon.
2types:
 Keratan sulfate І –cornea
 Keratan sulfate ІІ –skeletalmuscle
Function :
 Maintains the corneal transparency.
12
0

122. Heteropolysaccharide
12
2
• Agar :
Contains galactose , glucose & othersugars.
Cannot be digested by bacteria.
So used as supporting agent to culture bacterial
colonies.
Also as support medium of immuno
diffusion &immuno-electrophoresis.
• Agarose :
galactose & 3,6 anhydro galactose units
Used as matrix for electrophoresis.