The document discusses the structural organization of proteins, highlighting four levels: primary, secondary, tertiary, and quaternary. It explains the significance of amino acid sequences in determining protein function and the various bonds involved in protein structure, including peptide and disulfide bonds. Additionally, it details techniques for determining protein structure and functions of biologically important peptides.

1. Dr. N. Sivaranjani , MD
Asst. Prof.

2. Proteins are the polymers of L-
α amino acids.
Native protein structure is
essential for the biological
function of a protein
Levels of structural
organization of proteins :
Primary structure
Secondary structure
Tertiary structure
Quaternary structure
Loss of structure results in loss
of biological function

3. 4 Levels of Protein
Organization
1. Primary structure: The linear sequence of
amino acids forming the backbone of
proteins.
2. Secondary structure: The spatial
arrangement of protein by twisting of the
polypeptide chain.
3. Tertiary structure: The three
dimensional structure of a functional
protein.
4.Quaternary structure: proteins are
composed of two or more polypeptide chains
referred to as subunits. The spatial
arrangement of these subunits.

4. Proteins are formed of A.A. linked together by the following types of bonds
Covalent
bonds
Non Covalent
bonds
Peptide
bond
Disulfide
bond
Hydrogen bond
Electrostatic bond / ionic
interactions
Van der Waals interactions
Hydrophobic interactions
Strong bonds in protein structure

5. Covalent bond
Disulfide bond
Peptide bond

6. Non-covalent Bonds in Proteins

7. Primary structure
The Number and Linear sequence of amino acids in a protein is called the
primary structure.
Presence of specific amino acids at a specific number is very significant
for a particular function of a protein.
 Every polypeptide chain has unique a.a sequence, dictated by genes.
 Genetic diseases – proteins with abnormal amino acid sequences
 Bonds -- only covalent bonds – Peptide bond , disulfide bonds

8. Peptide bond formation
• α-COOH group of one a.a (with side chain R1) forms a covalent
peptide bond with α-NH2 group of another a.a ( with the side chain R2)
by removal of a molecule of water.
• It result in Dipeptide ( i.e. Two amino acids linked by one peptide
bond).
• Tripeptide (3 a.a linked by 2 peptide bond)
• < 50 a.a - “Peptides”,
• > 50 a.a - “Proteins”

9. Formation of Peptide Bond
Amino acid 1 Amino acid 2
H2O
H
H
H N
H
C
R
O
C
O
H
H
H N
H
C
R
O
C
H
C
R
C
O
H
H N
H
O
+
Amino gr Carboxyl gr
H
C
R
C
O
N
H
O
N terminal C terminal
Peptide bond

10. Naming of Peptide bond
H3N-Glutamate – cysteine - methionine – glycine-COO
N- terminal C- terminal
Glu – cys – met- Gly
glutamyl – cystenyl – methionyl-glycine
Amino acid sequence is read from N-terminal to C-terminal.
Protein synthesis occurs in the same way.
N-terminal a.a – “ yl” ; C- terminal a.a – “ine” .

11. Characteristics of peptide bond
• Partial double bond character (distance is 1.32Å which
is midway b/w single bond 1.49 Å and double bond 1.27Å)
• Rigid and planar
• Always in Trans configuration
• Side chains are free to rotate on either side of the
peptide bond
• The angles of rotation known as Ramachandran angle – determine
the spatial orientation of the peptide chain
Dr. GN Ramachandran

12. Importance of Primary structure
• Higher levels of organization of Proteins are dependent on the primary
structure
• Even a single amino acid change (mutation) in the linear sequence – cause
disease
• Eg:- Sickle Cell Anemia
HbA (normal Hemoglobin) the
6th amino acid in the beta chain
is glutamic acid - it is changed
to valine in HbS (sickle cell
anemia)

13. 2 Interchain disulphide bonds –
A chain 7th cysteine and B chain 7th cysteine
A chain 20th cysteine and B chain 19th cysteine
1 Intrachain disulphide bond -
between 6th and 11th cysteine residues of A chain
86 amino acids C-peptide
33
21
30
51 amino acids

14. Determination of Primary structure of Protein
Identification of amino acids with regard to their quality,
quantity and sequence in a protein structure

15. • Steps for Determining the Primary Structure
Steps Technique
I. Determination of amino acid composition in a protein
Complete hydrolysis of protein Pronase enzyme
Separation and estimation of amino acids Chromatographic technique
II. Degradation of protein into smaller fragments
Liberation of polypeptides Urea or guanidine hydrochloride
Determination of the number of polypeptide
Dansyl chloride
chains in a protein
Breakdown of polypeptides into fragments Enzymatic cleavage
Chemical cleavage
III. Determination of amino acid sequence Sanger's reagent
Edman's reagent
Nowadays, DNA sequencing is used to determine the amino acid sequence.

16. Determination of amino acid composition
1. Hydrolysis of protein
• Pronase- mixture of non-specific proteolytic enzymes that causes
complete hydrolysis of proteins.
2. Separation and estimation of amino acids:
 The mixture of a.a`s liberated by protein hydrolysis can be determined
by chromatographic techniques , by using Ninhydrin.

17. Degradation of protein or polypeptide into smaller fragments
• Liberation of polypeptides
• Urea or Guanidine hydrochloride - disrupts the non-covalent bonds and
dissociates the protein into polypeptide units.
• Disulfide linkages b/w the polypeptide broken- Performic acid
• Number of polypeptides
• Dansyl chloride (1-dimethyl amino naphthalene 5-sulfonyl chloride) –
Specifically binds with N-terminal amino acids to form dansyl polypeptides
which on hydrolysis yield N-terminal dansyl amino acid.
• Number of Dansyl AA = Number of Polypeptide chains in a Protein

18. Breakdown of polypeptides into fragments
• Polypeptides are degraded into smaller peptides - enzymatic or chemical
methods
• Enzymatic cleavage: exhibit specificity in cleaving the peptide bonds
• Chemical cleavage: Cyanogen bromide (CNBr ) commonly used
• Attacks Carbonyl side contributed by Methionine AA.
Enzymes Specific action
N terminal AA is identified by
Trypsin -commonly used Lysine and Arginine
Chymotrypsin Phe,Tyr, Trp, or Leu
C-terminal AA is identified by
Carboxypeptidase A Acidic / neutral AA
Carboxypeptidase B Proline

19. Determination of the amino acid sequence
• Sangers reagent: 1-fluoro-2,4-dinitrobenzene (FDNB)
• FDNB specifically binds with N-terminal amino acids to form a
Dinitrophenyl (DNP) derivative of peptide
• This is on hydrolysis yields DNP – amino acids ( N-terminal) and free
amino acids from the rest of the peptide chain.
• Used for identification of N terminal AA
• DNP-AA – identified by Chromatography

20.  Edmans reagent: Phenyl isothiocyanate
 Reacts with N-terminal amino acid of peptide to form a phenyl
thiocarbamyl derivative
 Phenyl thiohydantoin (PTH)-amino acid is liberated - Identified by
chromatography
Edman's reagent would then react with the second amino acid which now
has the alpha amino group
 Useful in sequencing first 10-30 amino acids
Label first AA
Release of 1st AA
Label 2nd AA
Polypeptide chain
Release of 2nd AA

21. For identifying the amino-terminal residue
The Edman’s degradation
Reveals the entire sequence
of a peptide
Sanger’s degradation

22. Sequenator
• This is an automatic machine to determine the amino acid sequence in a
polypeptide
• It is based on the principle of Edmans degradation .
• Amino acids are determined sequentially from N-terminal end
• The PTH-amino acid liberated is identified by HPLC.
• Sequenator takes about 2 hours to determine each amino acid.

23. Reverse sequencing technique
• It is the genetic material (DNA) which ultimately determines the sequence
of amino acids in a polypeptide chain
• By analyzing the nucleotide sequence of DNA that codes for protein, it is
possible to translate the nucleotide sequence into amino acid sequence.
• Demerits - fails to identify the disulfide bonds and changes that occur in
the amino acids after the protein is synthesized.

24. Secondary structure
• The spatial arrangement of protein by twisting and folding of
the polypeptide chain is called Secondary structure.
• The amino acids are located close to each other in their
sequence.
• Two types of secondary structures :- α-Helix and β-sheet

25. Alpha helix
• Most common and Stable conformation
• Spiral structure - Polypeptide bonds form
the back-bone and Side chain extend
outwards
• Stabilized by H bonding b/w carbonyl
oxygen and amide hydrogen.
• Right handed helix
• Amino acids per turn – 3.6
• Hemoglobin, Myoglobin -Abundant ,
Chymotrypsin – absent
• Proline – disrupts the α-helix
H bonding
The hydrogen bonds are individually weak but collectively,
strong enough to stabilize the helix

26. β-pleated sheet (β-sheets)
• The surfaces of β – sheets appear “pleated”, and these
structures are, therefore, often called “ β – pleated
sheets”.
• Polypeptide chains – fully extended
• distance between adjacent a.a -3.5Å
• Stabilized by hydrogen bonds
2 types
Parallel Anti -Parallel
strands in a sheet run in an opp.
direction - Silk fibroin
C
C N
C C
N
run in the same direction
– Flavodoxin
Carbonic anhydrase – contains both
N
N

27. Antiparallel
Parallel

28. • -helix – protein turns like a
spiral
• Keratin, fibrous proteins
structures are almost
entirely alpha helical (hair,
nails, horns)
-sheet – protein
extended –Globular
protein

29. Super secondary structures
• Some of the common structural motifs combining α – helices and β
– sheets
• found in globular proteins
These are :-
• β- α- β unit
• Greek key
• β-meander
Beta barrel
beta-alpha-beta motif Greek key motif β-meander motif

30. Triple helix
Collagen

32. Tertiary structure
• Peptide chain with its secondary structure ,may be further folded &
twisted about itself forming 3D arrangement of a functional
protein.
• It is a compact structure - hydrophobic side chains held interior
,hydrophilic groups - surface of the protein molecule.
• Vander Waal's forces , disulfide bonds(-S-S ), electrostatic bonds
and hydrophobic interactions contribute to the stability of tertiary
structure of proteins.
The function of a protein depends on its tertiary structure. If
this is disrupted, it loses its activity
Eg:- active / catalytic site of an enzyme

33. Domains
• A domain represents compact functional unit of protein .
• A polypeptide with 200 amino acids normally consists of two or more
domains
• Part of protein that can fold into a stable structure independently
• Domains are usually connected with relatively flexible areas of protein
• Different domains can impart different functions to proteins

34. Chaperones / Hsp
 Chaperone proteins participate in the folding of proteins.
 The hsp 70 ( 70 – kDa heat shock protein ) family of chaperones
binds short segments of hydrophobic AAs in newly synthesized
polypeptides.
 Chaperones prevent aggregation of polypeptide chain thus giving a
chance for formation of secondary and tertiary structures.
 Neurological diseases result from altered protein folding -
Alzheimer's disease (β – Amyloid protein, a misfolded protein is
formed in brain)
Prion diseases (altered secondary –tertiary structure)

35. Quaternary structure
• Many proteins have single polypeptide chains – Monomeric proteins
• Proteins having more than one polypeptide chains have this structure –
Oligomeric proteins
• Each polypeptide chain is called subunits or monomers.
• Subunits either function independently or may work co-operateively (Hb)
• Dimer – 2 polypeptide chains, Trimer , Tetramer – 3,4 polypeptide chains.
• The protein function is lost if subunits dissociate
• Bonds- non covalent interactions

36. • Hemoglobin
• 2  globin subunits
• 2  globin subunits
Other Examples:–
Immunoglobulin – 2 heavy chains and 2 light chains
Lactate Dehydrogenase (LDH) - Tetramer
Creatine Kinase (CK) - Dimer

37. • Study of Higher Levels of Protein Structure
• The higher levels of protein structure may be
studied by techniques using :-
• X-ray diffraction,
• Ultraviolet light spectroscopy,
• Optical rotatory dispersion,
• Circular dichroism,
• Nuclear Magnetic resonance (NMR).

38. Structure-Function Relationship
• The functions of proteins are maintained because of their ability to
recognize and interact with a variety of molecules.
Three dimensional structural conformation provides and maintains the
functional characteristics
Three dimensional structure is dependent on the 1⁰ structure
Difference in the 1⁰ structure causes loss of protein function

39. Heme
Transport proteins
 Hemoglobin
 Hb has a quaternary structure with 2 α and 2β
 Tetrameric protein – each Monomer has a Heme
unit which binds the oxygen
 Binding of oxygen to one Heme facilitates oxygen binding by other subunits
 In Primary structure any one amino acid replacement by another can cause
abnormalities in its function.
Eg:- Sickle cell Anemia :- In 6th position of
β-chain of Hemoglobin GLUTAMIC ACID is replaced
by VALINE.

40.  Structural proteins
 Collagen
Main fibrous component of skin, bone, tendon,
cartilage and teeth.
Triple helix – every 3rd a.a is Glycine.
 Proline, Lysine, Hydroxyl Proline, hydroxyl Lysine
Strength of collagen depends upon its structure
(triple helix)
In vitamin C deficiency - Collagen becomes weak as hydroxylation of
proline and lysine does not occur.
This causes reduced H-bonding and consequently weakness of collagen.

41. Enzymes
Enzyme catalysis needs precise binding of the
substrate to the active site of the enzymes.
This depends on structural conformation of the
active sites - precisely oriented for substrate
binding.

42. Biologically important peptides
Peptide name and the No of
amino acids
Origin Functions
Glucagon - 29 amino acids α cells of Pancreas Increases Blood glucose
Adrenocorticotropic hormone
– 39 a.a
Hypothalamus Stimulate and secrete the steroid
hormone corticotropin by anterior
pituitary gland
Oxytocin - 9 a.a Posterior pituitary Stimulates uterine contractions
Vasopressin – 9 a.a Posterior pituitary Increases blood pressure and has an
antidiuretic action
Angiotensin II – 8 a.a Liver Stimulates the release of aldosterone
from adrenal gland
ACE inhibitors – used in Rx of HTN
Glutathione (GSH) -3 a.a Antioxidant

43. Biologically important peptides
 When 10 or less number of amino acids are joined together, it is
called an Oligopeptide -Some of them are biologically active
 Glutathione, TRH, Encephalins, Oxytocin , Vasopressin, Angiotensin
I & II, Gramicidin S.
 Polypeptide hormones (more than 10 amino acids) – Insulin ,
Glucagon , Growth hormone, Gastrin, ACTH etc.

44. Glutathione (GSH)
GSH is a Tripeptide ( gama glutamyl – cysteinyl – glycine)
Gluamate – Cysteine - Glycine
SH
Reduced glutathione (GSH) –
active form
Oxidized glutathione (G-S-S-G)
– inactive form
Functions of Glutathione
• Membrane stabilization - Reduced glutathione is essential for
maintaining the normal structure of RBCs.

45. Biological antioxidants – it suppresses H2O2 and organic peroxides,
(lipid peroxides) to H2O by Glutathione Peroxidase.
Maintenance of biological activity of protein – helps to keep E in
active site
Prevention of Met Hb – keeps Fe in Ferrous state in Hb
Role in detoxification - Glutathione plays a key role in
detoxification of compounds by conjugation
Absorption of amino acids – small intestinal mucosal cell and
proximal renal tubules - ɣ glutamyl cycle
Disorder – Hemolytic Anemia

46. Thyrotropin releasing hormone (TRH) – 3 amino acids
Hypothalamus
PGA-Glu-His-Pro-NH2
Glu is modified to form pyroglutamic acid (cyclization of N
terminal Glutamate).
Proline – carboxyl gr of proline is amidated
It stimulates the release of TSH from antr. pituitary

47. Nanopeptide
Oxytocin – 9 amino acids Posterior pituitary
It stimulates uterine contractions.
Vasopressin / Anti diuretic hormone
– 9 amino acids
Posterior pituitary
It facilitate water reabsorption in DCT and Collecting ducts of
kidney – decreasing the Urine output.
Diabetes insipidus – decreased ADH synthesis

48. Pentapeptide
Enkephalins – 5 amino acids
Met-enkephalin - Tyr-Gly-Gly-Phe-Met.
Leu-enkephalin - Tyr-Gly-Gly-Phe-Leu.
CNS
Analgesic action – dec. pain

49. Angiotensinogen – 400 a.a Synthesized in Liver
Renin (Kidney)
Angiotensin I (10 amino acids)
Angiotensin Converting Enzyme (ACE)
Angiotensin II (8 amino acids)
 It has hypertensive effect – inc. BP
 Vasoconstriction
 Stimulates the release of
aldosterone from adrenal medulla
 Na retention
Octapeptide
Lungs
ACE inhibitors –
Captopril, Enalapril
are used in the Rx of
Hypertension

50.  Carnosine
 Dipeptide – βAlanine – Histidine
 Activates myosine ATPase activity – muscle contraction
 Chelates Cu2+ and facilitates absorption of Cu
 Aspartame
 Dipeptide – Aspartate and Phenylalanine
 Artificial sweetner

51. Primary
Secondary
Tertiary
Quaternary
S T R U C T U R E
P R O C E S S
Linear 1D
sequence of AAs
Folding 2D
Packing 3D
Interaction of
2 or more
subunits