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Structure function relationship of clinically important peptides
Structure-function relationship of clinically important Peptides
Dr. Rohini C Sane
Protein structure-function relationship
• The overall conformation of a protein , the particular position of amino acid
side chains in three-dimensional space determines the function of the
• The diversity of protein structure and its correlation with function can be
explained by the following clinically important proteins.
Carbonic anhydrase Formation and degradation of carbonic acid
Myoglobin(Mb) Stores oxygen in muscle cells
Hemoglobin (Hb) Transports oxygen from lungs to the tissue
Collagen Providestensile strengthtomanytissueinthebody
Elastin(withrubberlikeproperties) Occur in distensible structure
Quaternary structure of enzyme regulates its functions
Structural conformation of the active site the
enzyme precisely oriented for the substrate binding
Binding of enzyme to the substrate .
enzyme catalysis →product + enzyme set free for
substrate binding again
Disordered region of enzyme and its Importance
Many Disordered region become ordered region when a specific ligand is bound .
Disordered region gives flexibility and performs a vital biological role.
Disordered regions of
enzyme- catalytic site
ordered regions of
Substrate added to
set free at the end of
Enzyme-substrate complex –induced fit
❖Function of Carbonic anhydrase : catalyzes the reversible hydration of carbon
H2O + CO2 H2CO3(Carbonic acid)
1.H2O binds to Zinc ion (located in deep cleft of Carbonic anhydrase and is
coordinated to Histidine residue present at its active site).
Binding of CO2 to histidine residue is close to Zinc ion.
2.H2O→ H+ + OH- (Ionization of water to hydroxyl ion)
3.OH- +CO2(located proximally to OH- ions)→HCO-
Carbonic anhydrase facilitates the precise positioning of CO2 molecule and hydroxyl
ion OH- for the formation of bicarbonate ion HCO-
3 + H+ → H2CO3(Carbonic acid)
Thus , two substrates CO2 and H2O are brought in close proximity for reaction to
proceed and to facilitate the function of Carbonic anhydrase .
1.H2O binds to Zinc ion (located in a deep cleft of Carbonic anhydrase and is coordinated to
Histidine residue present at an active site of enzyme )
2.H2O→ H+ + OH- (Ionization of water)
3. Binding of CO2 toHistidine residue close to Zinc ion of Carbonic anhydrase
4.OH- + CO2( located proximally to OH- ions)→HCO-
3 + H+ →H2CO3(Carbonic acid)
Tertiary Structure of Hemoglobin (Hb)
Functions of Myoglobin
❖Functions of Myoglobin(Mb): acts as storage and transport protein for oxygen
in skeletal&cardiacmuscles. In resting muscle cells ,myoglobin binds oxygen
(O2)that has been released by Hemoglobin(Hb) under the conditions of
oxygen deprivation (in severe exercise for use O2 by muscle).
❖HbO2 (oxyhemoglobin) → → → Mb(Myoglobin) →MbO2(oxymyoglobin)→
tissue → Respiration
Oxygen (O2) transport
Oxygen dissociation curve of Myoglobin and hemoglobin
When the amount oxygen (O2)bound to the
Hemoglobin and Myoglobin is plotted
against the partial pressure of oxygen (pO2),
a hyperbolic curve is obtained for
Myoglobin whereas that for Hemoglobin is
When the (pO2) is high both Myoglobin and
Hemoglobin are saturated with oxygen.
At lower levels of (pO2), however ,
Myoglobin contains more oxygen O2 than
Bohr’s effect ,co-operative effect and 2,3
BPG effect are absent for Myoglobin .
Mb has higher affinity for Oxygen (O2) than Hb.
At PO2 in tissue (30mmHg): Mb is 90% saturated, Hb is 50%
saturated . Physical exercise: PO2 in tissue is (5mmHg), when
Mb releases all bound Oxygen (O2).
Clinical aspects associated with Myoglobin
❖Myoglobin : under physiological conditions , Myoglobin( being a small
molecular weight protein)is filtered and excreted in urine.
• is a sensitive maker for muscleinjury,makingitpotential cardiacmarkerfor
• rises rapidly after the chest pain at about the same rate as CK-MB.
• is non-specific ,since it is raised following any form of muscle damage.
• Estimation ofMyoglobin–usingmonoclonalantibodyforbyRIA/ELIZA/
• Cost of the analysis has prevented its widespread use.
• The cellular release Myoglobin is often associated with an increase in Creatine
kinase (CPK), Aldolase , Lactate dehydrogenase(LDH) , Serum Glutamate
Pyruvate Transaminase(SGPT) in patients with myocardial infarction.
❖Myoglobinuria : under physiological conditions , Myoglobin being low
molecular weight protein, it is filtered and excreted in urine. Presence of
excessive Myoglobin in the urine usually associated with muscle destruction
or rhabdomyolysis . Urine color becomes dark brown.
❖Damage to muscle( crush injury), releases Myoglobin to the circulation and
filtered by the kidney. If too much of Myoglobin is released into circulation ,
Myoglobin precipitate and obstruct the renal filtration system results in
acute renal failure.
❖Myoglobin is released from myocardium during Myocardial infarction (MI)
and is elevated in serum(cardiac marker)followed by its excretion in urine.
Quaternary structure of Hemoglobin
4 Polypeptides chains
( tetramer) associated by
non-covalent bonds :
2 Alpha() chains
2 Beta( )chains
It possesses Quaternary
Each chain/subunit has one heme
group and so one Fe2+ ion similar
to that of myoglobin.
Hb is found
exclusively in RBC
Structure of normal Hemoglobin
Hemoglobin variant Structure of normal Hemoglobin Abbreviation
Hemoglobin (HbA 1) 2 Alpha() chains and 2 Beta( )chains 22
Hemoglobin (HbA 2) 2 Alpha() chains and 2 delta( )chains 22
Fetal Hemoglobin (HbF) 2 Alpha() chains and 2 gamma ( )chains 22
Functions of Hemoglobin
❖Functions of Hemoglobin:
• Hemoglobin is found exclusively in RBC .It transports four molecules
oxygen(O2) from lungs(high pO2) to the tissues (low pO2) . Myoglobin is still
very saturated with oxygen at the (pO2) of tissue. It can transport H + and
carbon dioxide (CO2) from peripheral tissues to lungs for its excretion in air.
Deoxyhemoglobin + 4O2 oxyhemoglobin (in lungs)
Deoxygenation is the reverse process by which O2 is liberated.
Oxyhemoglobin 4O2+ Deoxyhemoglobin
• Hemoglobin is rich in amino acid Histidine ,helps to maintain pH of blood (acts
• Transports oxygen(O2)by Hb is regulated by 2-3-bisphosphate
Functions of Hemoglobin as a transporter of oxygen(O2) from lungs(high pO2) to the tissues
Quaternary structure of Hemoglobin favors its functions
❖ Hemoglobin found exclusively in red cells and functions as transporter of
oxygen(O2) from lungs to tissue . Each molecule carry four
oxygen(O2)molecules from lungs to the cells .This function is favored by
presence of one heme unit one each in monomer (4 heme→4 oxygen(O2)/
➢Binding of oxygen(O2) to one heme unit of tetramer facilitates oxygen
binding by other subunits. Each subunit has a heme binding pocket similar to
that of myoglobin .
➢It can transport H+ and CO2 from the tissue to the lung.
➢Binding of H+ and CO2 promotes release of oxygen(O2) from hemoglobin .
➢This allosteric interaction is physiologically important and as Bohr’s effect .
➢Even a single amino acid substitution alters the structure and thereby
Co-operative binding of oxygen to hemoglobin
• Binding of oxygen to Heme will increase binding of oxygen to other heme
• Affinity of oxygen for hemoglobin
• Last oxygen binds with affinity 100 time greater than first oxygen
• Heme –Heme interaction ( co-operative binding of oxygen to Heme )
• Release oxygen from one Heme will release oxygen from other
• As there is communication between Heme groups of hemoglobin
• Myoglobin is reservoir (transient )& supplier of oxygen
Oxygen concentration high Oxygen concentration low
Oxygen binds to
Oxygen is released to tissue
Structural changes in hemoglobin on oxygen binding
• Studied using x ray crystallographic
• Homotropic effect→ binding of oxygen to hemoglobin
• Heterotrophic effect → binding of 2,3BPG to hemoglobin
• Distance between two Beta-chain decreases oxygenation from 4nm to 2nm
• Increasing affinity for oxygen with addition every molecule of oxygen
• On oxygenation iron moves in plane of Heme
• Decrease in diameter of iron (movement of iron accompanied by pulling of
• affinity of hemoglobin for last oxygen > first oxygen ( 100 times greater )
• Cooperative binding of oxygen to hemoglobin or Heme →Heme interaction
• Release of oxygen from one Heme → Release of oxygen from other Heme
❖Therefore communication between Heme groups of hemoglobin.
On oxygenation iron moves in plane of Heme → decrease in diameter of Iron
→movement of Fe Is accompanied by pulling of proximal site →primary event of Heme
–Heme interaction of Hemoglobin
Structural changes in Hemoglobin on its Oxygenation and deoxygenation
Deoxy –Hb Hb O₂ Hb O₄ Hb O₆ Hb O₈
T form ↓ ↓ ↓ ↓ ↓
Oxygenation of hemoglobin
Quaternary structure of Hemoglobin favors its functions :Binding of oxygen(O2) to
one heme unit facilitates oxygen binding by other subunits.
Structural changes in Hb on oxygen binding
• Structural change in one subunit of hemoglobin on oxygenation is
communicated to other subunits
• Binding of oxygen to one Heme distorts globin chain to which it is
attached → distortion in neighboring chain →oxygen binds more
Effect of 2,3 BPG on oxygen affinity of Hb
• 2,3 BPG :Most abundant phosphate in RBC
• Molar concentration of 2,3 BPG = Molar concentration of Hb
• Synthesis ( synthesis through Rapport Leu Bering cycle)
2, 3 BPG mutase ( Glycolysis )
1,3 BPG 2,3 BPG
• Reinhold's & Ruth Benesch’s (1967 )→ 2,3 BPG decreases affinity of Oxygen to
• 2,3 BPG regulates the binding of oxygen
• 1mole of 2,3 BPG binds to 1mole of Deoxy Hb not to oxy –Hb
• Molecular concentration 2,3 BPG = Molecular concentration of hemoglobin
• At partial pressure of oxygen(O₂) in tissue: HbO₂+ 2,3 BPG→Hb 2,3 BPG +O₂
(release of O₂)
(Oxy–Hb) (De-oxy Hb)
• In tissue → 2,3 BPG shift curve towards right
1mole of 2,3 BPG binds to 1mole of Deoxy Hb not to oxy –Hb
Regulation of oxygen binding by 2,3 BPG
Effect of 2,3 BPG on oxygen affinity of Hb
2,3 BPG decreases affinity of Oxygen to Hemoglobin. In tissue → 2,3 BPG shift curve towards right
At partial pressure of O₂ in tissue: HbO₂+ 2,3 BPG→ Hb 2,3 BPG +O₂ (release of O₂)
(Oxy –Hb) (De-oxy Hb)
2,3 BPG shift oxygen dissociation curve of hemoglobin towards right to release of oxygen (O₂)
in hypoxic conditions ,anemia ,hyperthermia.
Effect of 2,3 BPG shift oxygen dissociation curve of hemoglobin
Clinical significance of 2,3 BPG:1
❖Function of 2,3BPG : Release of oxygen to tissue ( supply of oxygen to tissue )
to cope with oxygen demand → varied concentration of 2,3 BPG
1. Hypoxia : concentration of 2,3BPG in RBC increases during chronic hypoxic
a. Adaptation to high altitude
b. Obstruction to pulmonary odema ( air flow in bronchial blocked )
2. Anemia : concentration of 2,3 BPG in RBC increases in chronic anemic
conditions → to cope with oxygen (O₂) demand of body even at low Hb
Clinical significance of 2,3 BPG:2
3. Blood Transfusion : storage of blood in acid citrate dextrose → decrease in
concentration of 2,3, BPG ( O₂ remains bound to Hb )
• Blood stored in ACD fails to supply O₂ to tissue→ with 24-48 hr. until 2,3 BPG
• O₂ supply /tissue O₂ demand met adequately after 24-48 hrs.
• Blood with (ACD )+ Inosine ( Hypoxanthine Ribose )→ prevent decrease in 2,3, BPG
• Inosine→ phosphorylation of tissue→ entry into HMP shunt → get converted to
2,3BPG →increase in Conc in 2,3 BPG → release of oxygen
4.Fetal hemoglobin(HbF) : the binding of 2,3BPG to fetal hemoglobin is very weak.
Therefore, HbF has higher affinity for oxygen compared to adult hemoglobin (HbA).
This is needed for transfer of oxygen from the maternal blood to fetus.
Protein structure-function relationship in
Adult Hemoglobin(HbA1) and Fetal Hemoglobin (HbF)
• Function of Fetal Hemoglobin (HbF) : The transfer of oxygen from the
mother to the fetus.
• This function of Fetal Hemoglobin (HbF) is facilitated by structural
differences in between the hemoglobin molecule of the mother and that of
• Adult Hemoglobin(HbA1) : 2 Alpha() chains and 2 Beta()chains (22)
• Fetal Hemoglobin (HbF) : consist of 2 Alpha() chains and 2gamma ()
• The difference in amino acid composition between the beta chain of HbA1
and gamma ( )chains of (HbF) results in structural changes that causes
HbF to have lower affinity for 2,3- biphosphoglycerate (2,3 BPG) than HbA1
and thus greater affinity for oxygen.
➢Therefore , the oxygen released from the mother’s HbA1 is readily bound by
HbF in the fetus.
Protein structure-function relationship in Myoglobin and adult Hemoglobin(HbA1):1
Criteria Myoglobin Hemoglobin(HbA1)
myoglobin.Foursubunits of Hb
Structure of Myoglobin and adult Hemoglobin(HbA1)
chainhaving 153aminoacids(molecularweight16700)andonehemegroup(ironcontaining porphyrin).
Hyperboliccurve of oxygenbinding forMyoglobin
Sigmoidcurveof oxygenbinding forHemoglobin
At Highpartialpressureofoxygen(pO2)both myoglobin and hemoglobinaresaturatedwithoxygen.
Application : Myoglobin stillverysaturatedwith oxygenatthelowpartialpressureofoxygen(pO2)in
tissue facilitatingstoragefunctionintissue.Hemoglobinis aeffectivetransporterof oxygen,bindingwith
oxygeninthe lungswhere(pO2)ishighandreleasingitintissuewhere(pO2)islow .
Transport of oxygen by hemoglobin
PO₂ (mm) of Hg % saturation
Inspired air 158
Alveolar air 100
lung 90 97%
Capillary bed 40 60%
37% - 40% O ₂ release of oxygen at tissue level
Structural modification of hemoglobin by glycosylation and its application
• Glycosylated hemoglobin (Hb A1C): is formed by non –enzymatic reaction of
the aldehyde group of Glucose with amino terminal (N –terminal residue)
Valine of beta chains of HbA when blood Glucose enters the erythrocytes.
• Normal concentration of Glycosylated hemoglobin (Hb A1C): low ( to the
extent of 5% of total hemoglobin
• Formation of (Hb A1C) is proportional to blood glucose concentration.
• Glycosylated hemoglobin (Hb A1C) increases in Diabetes Mellitus : 12% or
more of total hemoglobin as serum glucose is high
• Since RBC have life span of 120 days , the content of Hb A1C is an indicator of
how effectively blood glucose levels have been regulated over the previous
2 or 3 months.
• Application of Glycosylated hemoglobin (Hb A1C) estimation : to follow the
effectiveness of treatment (management)for Diabetes Mellitus .
Glycosylated hemoglobin (Hb A1C)
Glycosylated hemoglobin (Hb
A1C): is formed by non- enzymatic
reaction of HbA with Glucose when
blood Glucose enters the
Application of Glycosylated
hemoglobin (Hb A1c) estimation :
to follow the effectiveness of
treatment for Diabetes Mellitus
Primary structure of Normal protein determines biological functions
Unique amino acid sequence specified by genes in a Normal protein
Specific amino acid sequence→ confers specific 3 dimensional structure
Specific Function arises from conformation
Normal physiologically active protein e.g. Normal hemoglobin
Consequences of Altered primary Structure in Abnormal protein
Mutation→ altered genetic constitution ( base sequence of DNA)
Altered amino acid sequence→ Altered 3 dimensional structure(conformation)
Altered or loss of functions arises from Altered conformation of protein
Structure function relationship of proteins
• Unique amino acid sequence
specified by genes
• Specific amino acid sequence→
confers specific 3 dimensional
structure ( conformation )
• Specific Function arises from
• Normal physiologically active
protein e.g. Normal hemoglobin
• Mutation→ altered genetic
constitution / base sequence of
• Altered amino acid sequence
→Altered 3 dimensional structure
( conformation )
• Altered or loss of Function arises
from Altered conformation
• e.g. abnormal or physiologically
inactive protein→ disease
condition e.g. hemoglobinopathy
Altered primary Structure of Abnormal hemoglobin
Altered primary Structure leading anemia in
Sickle cell hemoglobin ( Hb S) no
change in amino acid sequence of
6 th amino acid in beta chain of HbA1 (Glutamic acid
replaced by Valine in HbS )
HbM (Methemoglobinemia ) Substitution of Tyrosine with Histidine
Hb Chesapeake Arginine is replaced by Leucine at 92th amino acid in
alpha chain of Hb A
Alpha( ) thalassemia ( normally the
rate of synthesis and chains
Deficiency or absence of alpha chains → HbA of fetus
has tetramer 4 or 4 chains ( enlargement of live
Beta ( )thalassemia ( more common
inherited disease than Alpha
Deficiency or absence of beta chains →hemoglobin
found in RBC are HbA2 ( 2 2) and HbF ( 2 2 )
• This substitution generates ‘A stick patch’ on the surface of the beta chain of
both oxygenated and deoxygenated HbS .
• On the surface of deoxygenated normal HbA and deoxygenated HbS , there
exists a complement to the sticky patch.
• When HbS is deoxygenated ,its sticky patches can bind to the complementary
patches on another deoxygenated HbS .
• Binding of large number of deoxygenated HbS causes polymerization of
deoxygenated HbS forming long fibrous precipitate that mechanically disturbs
the red cell→ sickle shaped→ casing lysis and anemia.
• This polymerization will not take place when HbS is in oxygenated form as in
arterial blood .
Molecular basis of Sickle cell anemia
Linus Pauling ( 1954 Noble prize ) reported abnormal electrophoretic mobility & peptide
mapping Glutamic acid ( sixth position on beta globin chain ) replaced by Valine (Recessive
Mutation ) Hb A & Hb F prevent sickling.
sixth amino acid in beta() chain of HbA1 (Glutamic acid )replaced by Valine in HbS
Altered primary Structure leading anemia in early life
Sickle cell disease
Binding of large number of deoxygenated HbS causes polymerization of deoxygenated HbS
forming long fibrous precipitate that mechanically disturbs the red cell→ sickle shaped→
casing lysis and anemia.
HbM (Methemoglobinemia )
❖HbM (Methemoglobinemia) :
Substitution of Tyrosine with Histidine on hemoglobin result in
oxidation of ferrous is to ferric in heme . Hemoglobin in blood is
oxidized to methemoglobin. This condition leads to
HbM (Methemoglobinemia) :Substitution of Tyrosine with Histidine
Substitution of Tyrosine with Histidine
oxidation of ferrous is to ferric in heme→
➢Substitution of Arginine by Leucine at 92th amino acid in alpha chain
of Hb A results in increased affinity of hemoglobin for oxygen and
does not release as much oxygen to the peripheral tissue as does
normal hemoglobin HbA .
➢This leads to tissue hypoxia and polycythemia ( increased number of
RBC per unit volume) in ode to meet the oxygen needs.
Quaternary structure of Aspartate trans carbamylase
Aspartate trans carbamylase : An allosteric enzyme with 2 subunits : catalytic
subunit (C) and the regulatory subunit (R)
Quaternary structure of Lactate dehydrogenase (LDH )
❖Quaternary structure of Lactate dehydrogenase (LDH ):
• two types of polypeptide chains :H and M type
• five isoenzymes :
1. LDH 1 →H4
2. LDH2 → H3M
3. LDH3→ H2M2
4. LDH 4→ HM3
5. LDH 5 →M4
Isoenzymes of Lactate dehydrogenase
Subunit composition of LDH1
isoenzymes in heart cells
favors conversion of Lactate
Subunit composition of LDH5
in muscle cells favors
conversion of Pyruvate to
Quaternary structure of Lactate dehydrogenase (LDH ): Tetramer with 2 types of
polypeptide chains →H and M type
Structure of Human Insulin
In 1953 , Frederick Sanger determined primary structure of Insulin ( a pancreatic protein
hormone ) and showed for the first time that a protein has a precisely defined amino acid
sequence ( primary structure.)
Insulin and Glucagon(Polypeptide Hormones)
Hormone secreted by pancreatic cells
Polypeptide and a Dimer with 51 amino acids
regulates glucose metabolism and Induces
Amino acid sequence varies in different
Hormone secreted by pancreatic cells
Polypeptide and monomer with 29 amino acids
regulates glucose metabolism and Induces
Amino acid sequence is same in all mammalian
Structure of Human Insulin
❖Structure of Human Insulin : described by Sanger(Noble-1955 )
❖Dipeptide (2 polypeptide chains)of insulin has 51 amino acids
A polypeptide chain : 21 amino acids
B polypeptide chain : 30 amino acids
➢inter chain disulphide bonds:
a. between cysteine residues ( 7th amino acid of A chain and 7th
amino acid of B chain )
b. between cysteine residues (20 th amino acid of A chain and 19 th
amino acid of B chain)
➢intra chain disulphide bond : between cysteine residues (6 th
amino acid of A chain with 11 th amino acid of B chain)
Structure of Human Insulin
Carboxy terminal end
A chain : Asparagine
B chain : Threonine
Amino terminal end
A chain : Glycine
B chain : Phenylalanine
A polypeptide chain :
21 amino acids
B polypeptide chain :
30 amino acids
intra chain disulphide bond : between
cysteine residues( 6 th amino acid of A
chain with 11 th amino acid of B chain)
inter chain disulphide bonds
Primary Structure of human Insulin
A chain H2N Gly Cys Cys Cys Cys Asn
1 6 7 11 20 21
B chain H2N Phe Cys Cys Thr
1 7 19 30
Pig insulin differs from human insulin in only one position ,30th amino acid is alanine instead of Threonine.
Insulin from other animals like cattle ,sheep, horse etc. differ from human insulin in having a different
sequence of amino acids in the positions 8-9-10 in A chain .
This minor altered sequence does not result inappreciable change biological activity.
Amino acid substitution in Primary Structure of human Insulin
8 9 10 Achainof insulin
Thr Ser Ile Human
Ala Ser Val Bovine
Thr Ser Ile Pig
Ala Gly Val Sheep
Thr Gly Ile Horse
Site of Insulin Biosynthesis
❖Amino acids form primary structure → definite function
❖Peptide < 10 amino acids
❖Polypeptide > 10 amino acids
Proinsulin(single polypeptide chain with 86 amino acids, molwt.9000)
Human Insulin ( dipeptide 51 amino acids : polypeptide chain A →21
amino acid and polypeptide chain B →30 amino acids held together b
interchain disulfide bridges molwt.5734 )
➢The gene for insulin synthesis : located on chromosome 11 in beta cells
of pancreatic cells.
Removal of signal sequence in endoplasmic reticulum
Removal of C-peptide in Golgi apparatus
Pre proinsulin ( single polypeptide chain ):
108 amino acids ,mol wt. 11500
Proinsulin ( single polypeptide
chain):86 amino acids ,mol wt. 9000
Insulin ( dipeptide chain ):51
amino acids ,mol wt. 5734
Biosynthesis of Insulin from Preproinsulin:1
The gene for insulin synthesis : located on chromosome 11 in beta cells of pancreatic cells.
Biosynthesis of Insulin from Preproinsulin:2
In the beta pancreatic cells , insulin and proinsulin combines with Zinc to form complexes.
In this form it is stored in the granules of the cytosol which is released in response to
various stimuli by exocytosis.
Pre proinsulin ( single polypeptide chain ) :108 amino acids ,mol wt. 11500
Proinsulin ( single polypeptide chain):86 amino acids ,mol wt. 9000
acids, mol wt. 5734
Structure function relationship of C–peptide and insulin
C – peptide
Single polypeptide chain with 86 amino acids
Beta cells of panaceas synthesize insulin as a prohormone – Proinsulin . Biologically active
insulin (a dipeptide) is formed by removal of C – peptide ( the central potion ) of proinsulin .
C–peptide and insulin are synthesized in equimolar concentration. It useful index for the
endogenous production of insulin.
Biochemical aspects of Collagen
❖Biochemical aspects of Collagen:
1. Most abundant fibrous protein(major macromolecules) in human body :70 kg
body weight→ 12-14 kg of total protein→5kg of Collagen (1/3 of total protein)
2. Main Component of : connective tissue, skin(70%) ,bone (90%) tendon(85%),
cartilage ,teeth and liver(4%)
3. Synthesized by fibroblast in connective tissue and osteoblasts in bone
4. Made up of small fibrils → tropocollagen( fundamental units ) containing 3
polypeptide chains each of them in left-handed helix with 3 amino acid per turn.
5. rich in Glycine and rare amino acids like hydroxyproline, hydroxylysine
6. Cysteine and Tryptophan absent
7. have a triple helical secondary structure and rich in helix destabilizing amino
acids (Glycine ,Proline and Hydroxyproline). These amino acids prevent the
formation of the usual - helical and - pleated structure. Instead it forms a triple
helical secondary structure.
Triple stranded helix Structure of Collagen
Made up of small fibrils → Tropocollagen( fundamental units ) containing 3
polypeptide chains each of them in left-handed helix with 3 amino acid per
Arrangements of collagen fibers in cartilage of bone
Types of Collagen
❖19 different Types of Collagen , composed of 30 distinct polypeptide
chains encoded by separate genes.
❖ Numbering for Types of Collagen: Roman numerals I, II, III….XIX
❖Structure of collagen types : in principle , all types of collagen are
triple helical structures . The triple helix may occur throughout the
molecule or only a part of the molecule.
❖Each one suited to performed specialized function in tissue
❖e.g. Collagen Type I →skin, Collagen Type II → bone
Most abundant types of collagen found in human tissue and their
Type of collagen Distribution Composition of triple helix
I Skin , bone, tendon , cornea 2 -1(I), -2(I)
II Articular cartilage, intervertebral disc,
III Fetal skin ,cardiovascular system,
IV Basement membrane 2 -1(IV), -2(IV)
V Placenta, Skin 2 -1(V), -2(V)
Structure of collagen Type 1
❖ Structure of collagen Type 1:
1. Triple stranded helical structure present throughout the collagen
2. Shape : rod-like molecule → 1.4 nm diameter and 300 nm length
3. Number of Amino acid residues : 1000 per for each polypeptide
chain (3000 /molecule)
4. Amino acid contribution : 1/3 rd of amino acids are Glycine (every
third amino acid in collagen is Glycine.
5. Repetitive amino acid sequence : (Gly – X –Y )n ,where X and Y
represent other amino acids
6. Proline and hydroxyproline : 100 per for each polypeptide chain
7. Function of Proline and hydroxyproline : confer rigidity to the
8. Collagen Fibril formation : Triple helical molecule of collagen
assemble to form elongated fibrils . It occurs by a quarter staggered
alignment i.e. each triple helix is displaced longitudinally from its
neighbor collagen molecule by about one-quarter of its length
9. Collagen Fiber formation : Collagen Fibrils assemble to form rod like
10. Strength of Collagen Fiber : contributed by covalent cross linking of
formed between Lysine and hydroxylysine and also between Proline
Collagen molecules in Collagen fibers
Triple helical molecule of
collagen assemble to
form elongated fibrils .
Triple stranded helical structure
present throughout the collagen
assemble to form
rod like fibers .
Repetitive amino acid sequence (Gly – X –Y )n
Proline and hydroxyproline confer rigidity to the collagen molecule
Arrangement of Tropocollagen molecules in collagen fibril
Heads of Tropocollagen molecules 64 nm Cross striations
Sections of Tropocollagen moleculeCollagen Fibril formation :
Triple helical molecule of collagen
assemble to form elongated fibrils .
It occurs by a quarter staggered
alignment i.e. each triple helix
is displaced longitudinally from
its neighbor collagen molecule
by about one-quarter of its length.
❖Tropocollagen : Subunits of Collagen
• Shape : rod shaped
• Length : 300nm
• Thickness : 1.4 nm
• Molecular weight : 300,000
• Constituent polypeptides: three helically interwind polypeptides
of equal length (each with 1000 amino acid residues)
• Primary structure of collagen : all 3 or two out of three chains
have identical in amino acid sequence. Rich in Glycine (35%)and
Alanine(11%) , Gly-Pro-X or Gly-Hpr-X or
• Repetitive amino acid sequence : (Gly – X –Y )n ,where X and Y
represent other amino acids
• Secondary structure of collagen : Each of three polypeptide
chains of tropocollagen is itself -helix. Proline and
hydroxyproline form bends in polypeptide chains that they are
not compatible with -helix structure.
❖Collagen fibril :
• Triple helical molecules are associated into Collagen fibrils.
• It consists of recurring polypeptide subunits called tropocollagen, arranged
head to tail in parallel bundles . The heads of the tropocollagen molecules are
staggered along the length of fibers ,accounting for the characteristic 64 nm
spacing of the cross striations in most collagens .
• A section of tropocollagen molecule shows the backbone of triple helix . Each
of three polypeptide chains of tropocollagen is itself -helix whose pitch and
spacing is determined by the rigid R group of the numerous Proline and
hydroxyproline residue .
• The gap between the end of one triple helix and the beginning of the next
where there is the deposition of hydroxyapatite crystals in bone formation.
Constituent amino acids of triple stranded helix Structure of Collagen
• ScvConstituent amino
acids of Collagen
% of total amino acids
Proline and hydroxy
Lysine and hydroxy
Triple helical secondary structure of Collagen
Forces stabilizing Triple helical secondary structure of Collagen
❖Forces stabilizing Triple helical secondary structure of Collagen:
1. Hydrogen bonds : three left-handed helices are bound together by
interchain hydrogen bonds.
2. Lysinonorleucine bond: covalent cross links both within and
between triple helical units further stabilize Collagen fibers.
3. Electrostatic interactions
4. Hydrophobic interactions
Covalent cross-links in Collagen fibers
• Strength of Collagen Fiber : contributed by covalent cross linking formed
between Lysine and hydroxylysine and also between Proline and
• Covalent cross links are formed both within and between triple helical units
further stabilize Collagen fibers.
• The degree of covalent cross-linking in Collagen molecule increases with age .
• In Elder individuals : skin, blood vessels (Collagen containing tissue) become
less elastic and more stiff → health complications
Skin :Collagen containing tissue
In Elder individuals , skin, blood vessels (Collagen containing tissue) become less elastic and
more stiff → health complications
Collagen and calcific aortic valve stenosis(CAVS)
Biosynthesis of collagen
❖Biosynthesis of collagen: collagen is an extracellular protein but synthesized as an
intracellular precursor molecule before becoming a mature collagen fibril.
• Site : fibroblast ,osteoblasts in bones , chondroblasts in cartilage, odontoblasts in teeth
• Cellular location : ribosomes in endoplasmic reticulum (ER)
• Precursor : preprocollagen (a single polypeptide chain) with leader peptide at amino
terminal 20000 MW and carboxy terminal 35000MW.Both are not present in mature
• Function of preprocollagen: contains a signal peptide which directs the protein to each
endoplasmic reticulum (ER)
• Synthesis of procollagen : from preprocollagen in (ER) after cleavage of a signal peptide
• Post transcriptional modification of procollagen : hydroxylation, glycosylation and
disulfide formation . Followed by its secretion in extracellular medium by the way of Golgi
• Synthesis of collagen in extracellular medium : from preprocollagen after action of
aminopeptidase and carboxypeptidase to remove terminal amino acids. This followed by
a spontaneous assembly of polypeptide chains to form triple helical structure (with 1000
amino acids each) of collagen .
Types of cross links in collagen
Aldol condensation Schiff base
Structural modification of Collagen during its Synthesis
Glycosylationloss of peptide potion from N-terminal and C-terminal
Each of the 3 chains is in a left handed
helix with 3 amino acids per turn.
3 Chains are further twisted in right handed way to give
cable like structure.
Hydroxylation of Proline and Lysine by Lysyl hydroxylase
and Proline hydroxylase in presence of vitamin C→
Cross linking of hydroxy proline and hydroxy lysine
Since vitamin C is required for collagen synthesis ,a connective tissue , there is a delay in
wound healing process in vitamin C deficiency.
Intracellular and extracellular alterations of Collagen during
Intracellular alterations of Collagen Extracellular alterations of Collagen
Hydroxylation of Proline and some
Formation of intra and interchain
Glycosylation of some of the
Oxidative deamination of epsilon amino
groups of Lysine and hydroxylysine
Formation of intrachain and interchain
disulphide bonds ,mainly in the carboxy
and amino terminal ends
Cleavage of 25-35 kD portions at both
carboxy and amino terminal ends
Formation of triple helix Formation of quarter staggered
Functions of Collagen
❖Functions of Collagen : triple helical molecules are associated into fibrils. There is
gap between the end of one triple helix and the beginning of the next where there
is deposition of hydroxyapatite crystals in bone formation.
1. Gives tensile strength, support and shape to tissue . To break a collagen fiber of 1
mm in diameter, a load of 10-40 kg is needed. In disease status tensile strength is
2. Contributes to proper alignment of cells ,which in turn help in cell proliferation
and their differentiation to different tissue and organs .
3. Collagen which is exposed in blood vessels contributes to thrombus formation.
❖Collagen can be converted to
a. gelatin by boiling by splitting off some amino acids .Gelatin is highly soluble and
easily digestible. It forms gel on cooling and is provided as diet for convalescents
and invalids. But it lacks essential amino acid Tryptophan.
b. a tough hard substance on treatment with tannic acid (tannic process)
Genetic aspects of Collagen Synthesis
❖Genetic aspects of Collagen Synthesis :
1. Complex process
2. Involves at least 30 genes in human
3. about 8 post –transcriptional modifications
4. Inherited diseases due to gene mutations linked with collagen synthesis:
a. Ehlers-Danlos syndrome
b. Alport syndrome
c. Osteogenesis imperfecta
d. Epidermolysis bullosa
Abnormalities associated with collagen synthesis
Disease Abnormalities associated with collagen synthesis
Inherited disorders characterized by hyperextensibility of skin and
abnormal tissue fragility , hypermobile and lax joints
Alport syndrome Defect in formation of type IV collagen fibers found in the basement
membrane of renal glomeruli→ hematuria and renal disease
Characterized by abnormal bone fragility due to deceased synthesis of
Epidermolysis bullosa due to alteration in in the structure of type VII collagen fibers→ skin
breaks and blister formation even with minor trauma
Scurvy Deficiency of vitamin C→ defective post translational modification of
collagen→ bleeding gums ,poor wound healing, subcutaneous
Lathyrism (disease of
bone deformities )
Types of Ehlers-Danlos syndrome
❖Types of Ehlers Danlos syndrome :
• Ehlers-Danlos syndrome type V: inherited deficiency of Lysyl oxidase(copper
requiring enzymes)→prevents cross-linking of collagen→ arterio-vascular and
• Ehlers-Danlos syndrome type VI: inherited deficiency of Lysyl hydroxylase
→abnormalities of the eye ,severe scoliosis (abnormal vertebral curvature)
and hyperextensibility of skin and joints.
• Ehlers-Danlos syndrome type VII: non-serving of procollagen as a substrate for
the procollagen amino protease →hip dislocation , increased skin elasticity
and short stature.
Ehlers-Danlos syndrome : Clinical manifestations
Hyperextensibility of skin and joints
❖An autosomal dominant trait.
❖Molecular basis : defect in the gene coding for fibrillin -1 located on
chromosome 15 → deficient of deposition of fibrillin -1 and elastin which are
components of microfibrils or defect in the gene coding for fibrillin -2 located
on chromosome 5 → deficient of deposition of fibrillin -2→congenital
❖Clinical manifestations of Marfan's syndrome :
a. Arachnodactyly (long digits)
b. Ectopia lentis(dislocation of lenses)
c. Hyperextension of joints
d. Aortic aneurism
Molecular basis of Marfan's syndrome
Molecular basis : defect in the gene coding for
fibrillin -1 located on chromosome 15 → deficient of
deposition of fibrillin -1 and elastin which are
components of microfibrils or defect in the gene
coding for fibrillin -2 located on chromosome 5 →
deficient of deposition of fibrillin -2→congenital
An autosomal dominant trait
Accumulation of Homocysteine
Reaction of Homocysteine with Lysyl aldehyde formed by Lysyl oxidase
Prevention of cross- linking of Lysine residues in connective tissue
Skeletal deformities ,vascular and ocular defects
❖Normal Homocysteine levels (blood): 5-15 micromoles/L
❖Hyperhomocysteinemia : homocysteine levels (blood) increased 50-100 times→
increased risk of coronary artery diseases ,urinary excretion of homocysteine
increases( >300 mg/24 hr.).
❖Causes of Hyperhomocysteinemia:
a. Vitamin B6 and/or B12 deficiency
c. Tobacco smokers
d. Alcoholics →chronic pancreatitis
e. Congenital diseases
f. Pre –eclampsia of Pregnancy
g. Elderly persons
❖Congenital Hyperhomocysteinemia : due Cystathionine beta-synthase deficiency
❖Clinical Signs and symptoms of Hyperhomocysteinemia :
a. Mental retardation
b. Charley Chaplin gait
c. Skeletal deformities
d. ocular defects: glaucoma myopia , Ectopia lentis(dislocation/ subluxation of
e. vascular defects: intravascular thrombosis
❖Molecular basis /changes : increased Homocysteine→ activation of Hageman’s
factor→ increased platelet adhesiveness → intravascular thrombosis→ life
❖Biochemical changes : increased serum Homocysteine and Methionine levels,
increased urinary excretion of Homocysteine( > 300mg/24 hr.), reduced plasma
Clinical manifestations of Congenital Hyperhomocysteinemia
Management of Hyperhomocysteinemia
❖Management of Hyperhomocysteinemia :
a. Dietary supplementation of Vitamin B6( 500mg /per day) and/or B12
b. Diet Low in Methionine and rich in Cysteine supplemented
❖Cyanide – nitroprusside Biochemical test for diagnosis (in urine): positive
➢Other diseases associated with Hyperhomocysteinemia: neurological
Protein structure-function relationship of Menke’s kinky hair syndrome
❖Menke’s kinky hair syndrome :
• An x-linked defect (affects only male child).
• Molecular basis :absence of an intracellular copper binding ATPase protein(mutation
in ATP7 A gene)→dietary copper absorbed from GI tract; but cannot be transported
to the blood .
• Copper that has entered into intestinal cells is not able to get out of the cell and so it
gets accumulated there . Therefore , Copper(a constituent of Lysyl oxidase)is not
available for metabolism ,resulting in defective cross linking in collagen molecule of
• Defective Vascular( weakening of walls of major blood vessels including aorta
→aneurysm→ fatal rupture of aorta→ cardiac failure) and connective tissues.
• Child dies in infancy.
➢Copper binding ATPase protein present in intestinal cells are different from that
present in liver and extrahepatic tissues . Therefore , Clinical manifestations of
Wilson’s disease and Menke’s disease are different .
Absence of an intracellular copper binding ATP ase protein( mutation in ATP7 A gene)
Accumulation of Copper in intestinal cells (Copper that has entered into is not able to get
out of the cell and so it accumulated gets there)
Unavailability of Copper for metabolism and function of Lysyl oxidase
Defective cross-linking in collagen molecule of connective tissue
Defective formation of Vascular and connective tissues
Death of the Child in infancy
Degradation of collagen by Collagenase
❖ Clinical applications of degradation
of Collagen by collagenase:
• Reabsorption of bone and cartilage
• Postpartum involution of uterus
• Paget’s disease
• Rheumatoid arthritis
• Gas gangrene
• Tumor metastasis
➢ Adult human tissue do not have
significant amount of collagenase
➢ Tissue collagenase is active in animals
whose tissue undergo a degree of
remolding e.g. tadpoles
❖ Gas gangrene:
• Collagenase produced by
Clostridium histolyticum splits
each polypeptide chain at the
site indicated ( X-Gly-Pro-Y)
• Connective tissue barriers
destroyed by bacterium
✓ Collagen: a protein resistant to
action of by ordinary
Clinical applications of degradation of Collagen structure by Collagenase
Gas gangrene : degradation of collagen
structure by collagenase by bacterium
Osteoarthritis and Rheumatoid Arthritis
1. A Connective tissue protein imparting high tensile strength
2. Occurrence : the major component in yellow elastic fibers of connective
tissue →lungs, elastic ligament , arterial blood vessels( especially large
vessels like aorta ,tendons
3. Formed in large amount in uterus during pregnancy
4. Are hydrolyzed by pancreatic elastase enzyme
Amino acid composition of Elastin
❖Tropoelastin(the basis subunit of elastin fibrils):
• contains about 800 amino acid residues
• Rich in non-polar amino acids such as Alanine, Leucine, Valine , Isoleucine
and Proline .
• Contain high amounts of Glycine, Proline(like collagen)
• One-third the residues are Glycine but No repeat sequence of (Gly-X-Y)n
• Less hydroxyproline
• Do not contain Cysteine, Methionine , Histidine , 5-hydroxylysine,
• No triple helix
Biosynthesis of Elastin
Biosynthesis : is synthesized as Tropoelastin
formation of hydroxyproline
➢Collagen has aldol cross links, while elastin has Desmosine cross links.
➢When elastin matures , Desmosine cross links are formed from lysine
➢Once elastin matures , elastin is very stable ,turn overrate is very low due to
different crosslinks .
Desmosine: Cross links of Elastin
❖Cross links of elastin:
• More complex than those in collagen.
• the major cross links in elastin are Desmosine.
• are formed from 4 Lysine residues. Some Lysine residue of Tropoelastin get
oxidized by lysine oxidase( copper containing enzyme) to aldehyde
derivative of lysine called Allysine.
3Allysine + unmodified Lysine →lysinonoleucine cross links of
Desmosine (by condensation)
• permit the elastin to stretch in two dimensions and subsequently recoil
during the performance of its physiologic functions.
• are destroyed by elastase .
✓Deficiency of alpha-trypsin (an inhibitor elastase)can result in Emphysema.
Emphysema: a clinical condition related to loss of Elastin function
Deficiency of alpha-trypsin (an inhibitor elastase)can result in emphysema.
Comparison of primary structure of Collagen and Elastin
Many different genetic type One genetic type
It has no capacity to stretch It has capacity to stretch and subsequently to
Formation of triple helix secondary structure No triple helix secondary structure
Presence of Hydroxylysine absence of Hydroxylysine
Presence of Glycosylated hydroxylysine absence of Glycosylated hydroxylysine
Formation of intramolecular aldol cross links Formation of intramolecular Desmosine cross
❖Human fibrinogen ( factor I):
1. Soluble glycoprotein
2. 2-3 % plasma protein(plasma fibrinogen
3. Consist of 6 polypeptide chains → two A ,two B
4. Structural formula= (A )2 (B )2 2
Structure –function relationship of fibrinogen
Fibrin monomer formation
Fibrin monomers stick together to form hard clot formation
Stabilization of clot formation by cross linking between Glutamine and Lysine
Red cells get entangled in fibrin clot → red color of clot
Proteolytic cleavage catalyzed Thrombin (IIa)→ Release of fibrinopeptides A and B
Biochemistry of Albumin
❖Biochemistry of Albumin:
• Plasma concentration : 3.5 - 5.5 gm/dl(60% of plasma proteins )
• Molecular weight : 69000
• Structure : a single polypeptide with 585 amino acids with 17 disulfide
• Site of synthesis : liver ( 12gm/day→25% of total hepatic protein)
• Half life : 20 days
• Application of Measurement of Plasma concentration→ Liver function test
Functions of Albumin (Globular proteins)
❖ Functions of Albumin :
1.Nutritive(serum albumin ,ovalbumin , Lactalbumin):
serves as source of amino acids for protein synthesis
particularly in nutritional deprivation of amino acids.
2. Transport :binds and transports plasma free fatty acids
,bilirubin, steroid hormones ,Calcium and Copper in
3. Buffering function : among the plasma proteins,
albumin has maximum buffering capacity(lower than
bicarbonate buffer system).
4.Osmotic function : due to high concentration and low
molecular weight .It plays predominant role in maintaining
blood/plasma volume and body fluid distribution. It
contributes to 75-80% of the total plasma osmotic
pressure(25 mm Hg) .
• The process of Protein folding is complex.
❖Causes of Protein misfolding :
2. Gene mutations
❖Consequences of Protein misfolding : misfolded protein usually get
degraded . However ,as the individual age progresses , misfolded protein
get accumulated and cause number of diseases.
❖Group of diseases due to Protein misfolding :
a. Prion diseases
Prion diseases due to Protein misfolding
❖Prion: represents proteinous infectious agents.
1. the altered forms of normal proteins.
2. No difference in the primary structure (amino acid sequence) and post-
translational modifications observed.
3. Certain changes in three –dimensional structure.
4. Major alterations is the replacement of alpha-helices by beta-sheets in PrP which
confers resistance to proteolytic digestion of Prions protein.
5. highly infectious agents and can act as template to convert non-infectious
proteins with alpha-helices to infectious form .
6. The process continues in exponential manner to accumulate a large number of
prion proteins in tissue.
Prion: represents proteinous infectious agents
Apolipoprotein E 2 (APO-E2) : responsible for production of chaperons of Tau protein→
the risk for Alzheimer's disease.
A model for the formation of infectious prions
Alpha-helix of a protein(non-infectious) Infectious prion (with beta –sheets}
Two molecules of Infectious prions (with beta-sheets)
These two molecules of Infectious prions separate and convent another two non-
infectious proteins to Infectious prions with beta –sheets.
Exponential increase in Infectious prions
A model for the formation of infectious prions
• Defects in mitochondrial genome will lead to myopathies. Leber’s Hereditary
Neuropathy (LHON)is caused by a single base mutation which alters one
Arginine to Histidine in NADH Coenzyme Q reductase.
OXPHOS(Oxidative phosphorylation)Disease Clinical features
Leber’s Hereditary Neuropathy (LHON) Complex I defect, blindness ,cardiac
Leigh’s syndrome Complex I defect , NDUFS gene defect,
Myoclonic epilepsy ragged red fiber disease
Myoclonic epilepsy, myopathy , dementia
Mitochondrial encephalopathy lactic acidosis
stroke like episodes ( MELAS)
Complex I defect , lactic acidosis, stroke ,
myopathy, seizures, dementia
❖Amyloidosis: refers to the altered proteins with beta sheets that accumulate in the body
particularly in the nervous system.
1. Extracellular Proteins found in tissue and body fluids resembling starch.
2. pathological deposit formed by protein misfolding or due to gene mutations associated
with a group of disorders collectively called amyloidosis.
3. pathological deposit exert pressure on the vital organs and eventually cause their death.
4. at least 15 different proteins found in a amyloidosis
5. are not infectious agents as prion proteins.
6. accumulate as the age advances (aging).
7. implicated in degenerative diseases ( e.g. Alzheimer'sdisease) and multiple myeloma.
8. Secondary amyloidosis associated with inflammatory or infectious diseases.
9. Familial amyloidosis: inherited genetic mutations
10. Diagnosis / Detection of amyloidosis: Amyloids + Congo red + polarized light→ Apple
Diseases associated with Amyloidosis due to Protein misfolding
Diseases Abnormal misfolded Protein
Alzheimer’s disease Beta amyloid
Cystic fibrosis CFTR
Parkinson’s disease Alpha synuclein
Huntington’s disease Huntingtin
Creutzfeldt Jakob disease Prion
• Three dimensional conformation of proteins important for biological
• some proteins can generate the functionally active conformation
spontaneously e.g. ribonuclease.
• Majority of attain correct conformation ,through assistance of certain
proteins called Chaperones.
Role of Chaperones in protein folding
Functions of Chaperones
❖Functions of Chaperones :
1. are heat shock proteins.
2. facilitate and favor interactions on the polypeptide surfaces to
finally give the specific conformation of a protein.
3. can reversibly bind to hydrophobic regions of unfolded proteins and
4. can stabilize intermediates and prevent the formation of incorrect
5. prevent undesirable interactions with other proteins.
6. these activities of chaperones help the protein to attain compact
and biologically active conformation.
Functions of Chaperones
Chaperones facilitate and favor interactions
on the polypeptide surfaces to finally give
the specific conformation of a protein.
Chaperones can reversibly bind to
hydrophobic regions of unfolded proteins
and folding intermediates.
Types of chaperones
❖Types of chaperones :
1. heat shock protein (Hsp)system : consist of 70 kDa heat shock
protein (Hsp 70) and 40 kDa heat shock protein (Hsp 40) . These
proteins can bind individually to the substrate protein and help in
formation of protein folding.
2. Chaperonin system : a large oligomeric assembly which forms a
structure into which the folded proteins are inserted. It mainly
consist of Hsp 60 and Hsp 10 i.e. 60 kDa and 10 kDa Hsp. They are
required at later part of protein folding process and work in
association with Hsp 70 system.
Chaperonin system : a Type of Chaperone
It is a large oligomeric assembly which forms a
structure into which the folded proteins are
It is required at later part of protein folding
process and work in association with Hsp 70
Heat shock protein (Hsp)system: a Type of Chaperone
Heat shock protein (Hsp)system : consist of 70 kDa heat shock protein (Hsp
70) and 40 kDa heat shock protein (Hsp 40) . These proteins can bind
individually to the substrate protein and help in formation of protein folding.
Protein misfolding and diseases
❖Protein misfolding and diseases :
The failure of a protein to fold properly and generally leads to its rapid
degradation. Prions (proteinous degraded infectious agents) are aggregates
of misfolded proteins or their partially degraded products . Prions exhibit
the characteristics of viral and microbial pathogens.
❖Protein misfolding and Cystic fibrosis (CF):
1. A common autosomal recessive disease
2. with mutations that result in abnormal protein cystic fibrosis
transmembrane conductance regulator (CFTR)
3. Mutated CFTR cannot fold properly ,not being able to get glycosylated
and transported . Therefore ,CTFR gets degraded.
❖Protein misfolding and neurological diseases :
Prion are implicated in Alzheimer’s disease , mad cow disease ,
Huntington’s disease, Creutzfeldt – Jacob disease
Cystic fibrosis (CF) and Alzheimer'sdiseasearedueProtein misfolding
The failure of a protein to fold properly and generally leads to its rapid
Alzheimer’s disease and chaperones
d. Personalitychangeswithabnormalbehavior→patients entervegetativestatewithno
Genes associated with Alzheimer's disease
Genes associated with Alzheimer's disease Located onChromosome
APO E4(Apolipoprotein E4) 19
Gene S 182 14
Alpha()secretase( proteolytic enzyme)
1. Expressed and present on cell surfaces(trans-membrane region).
2. A proteolytic enzyme.
3. Are members of ADAM(a Disintegrins and metalloprotease domains).
➢Secretase complex is a prime target for pharmacological interventions of AD.
Amyloid precursor protein(APP- a transmembrane protein )
Alpha ()secretase Beta ()secretase Gamma( )secretase
Soluble Alpha protein
insolubleproteins withAlpha()andbeta ()regions–toxic
Structure-functionrelationshipofAmyloid precursor protein(APP) in Alzheimer's
Criteria Normal Amyloid precursor protein( APP) Amyloid precursor protein
(APP) in Alzheimer's disease
Mutation of a gene coding for
APP→ substitution of Valine by
Solubility Soluble Insoluble(cannot be degraded by
Cathepsins and are deposited in
Substrate for Alpha ()secretase( proteolytic enzyme) Beta ()secretase( proteolytic
Soluble protein(sAPP) Insoluble beta- protein with 40-42
amino acids gets deposited →
Action of beta and gamma secretase on the Amyloid precursor protein(APP) can result in toxic
conformation , called alpha-beta peptide (40-42amino acids )with neurotoxic activity→
dementia of Alzheimer'sdisease.
Structure-functionrelationshipofBeta-Amyloid precursor protein(APP) in Down syndrome
Substitution of Valine by Isoleucine
Increased rate of biosynthesis of Beta-APP
Deposition of insoluble Amyloid plaques
sAPP and Alzheimer's disease
❖ sAPP : soluble form of Amyloid precursor protein results from its proteolytic cleavage
by -Secretase. It is released from neurons in response to Electrical activity.
❖Functions of sAPP in Modulation of :
1. Synaptic plasticity
3. Neuronal excitability
4. Neurite outgrowth
5. Neuronal Cell survival
C-Gmp (guanosine 3’,5’ –cyclic monophosphate )
Modulation of activities of K-channels
sAPP : a soluble neuroprotective protein APP with C-terminal end of truncated (s-APP ) +amyloid beta-
peptide(A –beta) with neurotoxic activity
-Secretase - secretase
Alzheimer's disease: is associated with mutation in secretase leading to decreased levels of sAPP protein and or
elevated levels of A-beta( APP )protein→ Dementia
amyloid beta peptide with neurotoxic activity
Regulation of Beta-Amyloid precursor protein(APP) synthesis by 25- hydroxy
➢27- hydroxy cholesterol(27OHC)→ regulate a number of key enzymes within
the brain→ regulate synthesis of Beta- Amyloid precursor protein(APP)→
prevent dementia/ Alzheimer's disease .
➢27- hydroxy cholesterol→ suppresses expression of gene responsible for
synthesis of Arc( a cytoskeletal-associated protein responsible for memory
➢Alzheimer's disease is associated with low levels of Arc.
Toxic effects of Beta-Amyloid precursor protein(APP) on neurofibrils
❖Toxic effects of Beta -Amyloid precursor protein(APP) on neurofibrils:
1. Beta -Amyloid precursor protein(APP) cause oxidative injury and changes
2. Changes in intracellular calcium homeostasis
3. Cytoskeletal recognition
➢The gene coding beta-APP is located on chromosome 21 .Therefore , in
Trisomy 21(Down syndrome) the rate of production of Beta-Amyloid precursor
protein(APP) is increased leading to early onset of Alzheimer’s in patients with
Pathogenesis of Alzheimer's disease and other Tauopathies
Self –assembly of tangles of paired helical
filaments and straight filaments
Enhanced activity of protein kinases and
diminished activity of phosphatases
Criteria Normal Tau proteins Tau proteins in Alzheimer's disease
Solubility Soluble and
Insoluble(cannot be degraded by
Cathepsins and are deposited around
Function Required for
channels in nerve
Loss stability of microtubules ,hyper
polymerization of tubulins in neurons
→deceased synthesis of Acetyl CoA→
Diabetes mellitus ,hypertension are
risk factors for Alzheimer's disease.
Tauopathies:Biochemical changes duetoTau proteins in Alzheimer's disease : 1. Threonine
/Serine kinases catalyze phosphorylation of Tau proteins → self-assembly of paired helical
filaments of Tau proteins → tangles of Tau→ disruption of microtubules in neurons of CNS →
Dementia 2.Changes in intracellular Calcium homeostasis .
Dementia :due to Mutation of a gene coding for
Transmembrane protein Presenilin-1 and Presenilin-2
Mutation of a gene coding for Transmembrane proteins
Presenilin-1 and Presenilin-2
Excessive production of Beta- Amyloid precursor protein(APP)
Deposition of insoluble Amyloid plaques in neurofibrils
Apolipoprotein E4 associated with Familial Alzheimer's disease
❖Apolipoprotein E : Arginine rich protein. It is present in chylomicrons, LDL, and VLDL . It is a ligand
for hepatic uptake. Normal Blood levels Apo E 2 mg/dL. Gene for Apolipoprotein E is polymeric and
located on chromosome 19 . It has has 3 alleles and therefore 6 possible combinations .
❖Liver and also Astrocytes make Apo-E. It is involved in cellular transport of lipids in CNS. Apo-E is
associated with lipoprotein glomerulopathy. Apo-E has four isoforms viz. ApoE- I, II, III, IV.
❖Two of these isoforms i.e. Apolipoprotein E- II and Apolipoprotein E- IV, increase the risk for
Alzheimer's disease by eight folds.
❖Apo E- IV is implicated in the development of senile dementia and Familial Alzheimer's disease
❖Familial Alzheimer's disease: 30% cases of (AD) with genetic background are associated with
Apolipoprotein E- IV .
➢Apolipoprotein E- II (Apo E -II) : responsible for production of chaperons of Tau protein→ the risk
for Alzheimer's disease.
Zinc deficiency and Alzheimer's disease
❖Zinc binds to beta amyloid to form plaque →dementia → Alzheimer's disease
Glutamate transporters in Alzheimer's disease
• L –Glutamate : an excitatory neurotransmitter in mammalian CNS.
• EAAT : high affinity glutamate transporters (5 types viz EAAT 1-5)
• Neural EAATs : play specialized roles at neural synapses.
• The transporters EAAT1 and EAAT2 present in glial neural cells and are
responsible for majority of Glutamate uptake.
• Dysfunction of EAAT is implicated in pathology of neurogenerative conditions
including Alzheimer's disease( other conditions : Huntington’s disease
,epilepsy , ischemic stroke, amyotrophic lateral sclerosis).
• Trafficking ,splicing, and post-transcriptional modifications of EAAT are
exploited in treatments of these conditions.
Clinical aspects of N-acetyl –D-aspartate receptors and Transcription factor NFKB
❖Genetic mutations or age-related changes in N-acetyl –D-aspartate receptors
and Transcription factor NFKB may promote neuronal degeneration indicated
in Alzheimer's disease by increasing production of A-beta and or decreasing
levels of neuroprotective s-APP-alpha .
Role of Glutathione in prevention of Alzheimer's disease
Alzheimer's disease: is due cellular accumulation of aggregates of misfolded proteins or
their partially degraded products . The term Prion ( proteinous infectious agents ) is coined
to collectively represent them. Reduced Glutathione prevents amyloid formation by
activating enzyme Glyoxalase and thus restores memory.
Diagnostic tests for Alzheimer's disease
❖Diagnostic tests for Alzheimer's disease
1. Elevated levels of Tau proteins in CSF
2. Elevated levels of soluble -Amyloid precursor protein Beta in CSF (sAPP)
3. Decreased levels of (s-APP)
4. Serum DHEA
➢APP-beta mutations are linked to some inherited forms of Alzheimer's
➢ s-APP is approximately 100-fold more potent than (s-APP) in protecting
hippocampal neurons against excitotoxicity.
Management of Alzheimer's disease
1. N –methyl aspartate an antagonist → slows the progression of disease
2. Omega-3-fatty acids → prevention disease
3. Long term anti-inflammatory drugs→ reduce incidence of disease
4. Beta() and Gamma ()secretase inhibitors
➢ Limitation of Gamma ()secretase inhibitor therapy : Gamma ()secretase is
required by variety of physiological substrates therefore ,there is a need to
develop substrate specific compounds.
Alpha-synuclein and Parkinson’s disease
• A degenerative disease –affecting muscular coordination.
• Two genes are associated with this disease
1. Gene for Alpha-synuclein is mutated in Parkinson’s disease. Alpha-
synuclein is found in Lewy bodies(=inclusion bodies) found in many regions
of the brain in this disease . Lewy bodies are responsible for neuronal
degeneration of Parkinson’s and Alzheimer’s disease.
2. Gene Codes for protein- Parkin which is associated with juvenile form of
❖Cystic Fibrosis (CF) is a multisystem disease that presents
• in neonates ,with failure to pass the first feces containing bile,
intestinal debris and mucus ( meconium ileus )
• in early childhood with respiratory infections
• in the adults
❖Inheritance of Cystic Fibrosis (CF) : autosomal recessive disorder
Pathogenesis of Cystic Fibrosis
❖Cystic Fibrosis arises due to mutations in gene located on
chromosome 7 encoding the Cystic Fibrosis transmembrane regulator
(CFTR ) protein that regulates transmembrane chloride transport .
❖ The most common mutation is F 508 mutation which refers to
deletion of three base pairs , resulting in the absence of Phenylalanine at
position 508 in amino acid sequence of CFTR .
Structure-functionrelationshipofCystic Fibrosis transmembrane regulator (CFTR )
❑Absence of Cystic Fibrosis transmembrane regulator (CFTR ) /chloride
channel leads to following Consequences :
• Exocrine pancreatic insufficiency with impaired secretion of sodium,
bicarbonates and water resulting in increased viscosity (mucoviscoidosis)
,obstruction of pancreatic duct , pancreatic fibrosis and obstruction of
• Chronic airways infection that affects mucus secretion in the bronchi with
recurrent respiratory infections ,bronchiectasis and chronic lung disease
• Malabsorption ,cirrhosis of liver and cholelithiasis due to defective secretion
of chloride and water
• abnormal sweat gland function due to excessive excretion of sodium and
chloride in sweat
• Abnormal urogenital functions
Diagnosis of Cystic Fibrosis
❖Diagnosis of Cystic Fibrosis is based on:
1. Clinical symptoms
2. Measurement of pilocarpine induced sweat electrolyte concentration:
3. Na ⁺ and Cl⁻ in sweat ( 70 mmols /L or mequ /L )
4. Neonatal screening test : increased plasma immunoreactive trypsin
5. Prenatal screening test : for F 508 mutation