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• The linear sequence of amino acids in a polypeptide.
• Determined by peptide bonds (–CO–NH–) which are strong, planar, and mostly trans.
• Sequence dictates higher-order folding and final protein function.
• Even a single amino-acid mutation (e.g., Val for Glu in sickle cell anemia) drastically changes structure and function.
Local folding patterns stabilized by hydrogen bonds between backbone –C=O and –NH groups.
• Right-handed spiral with 3.6 residues per turn.
• Stabilized by intrachain hydrogen bonds every 4th residue.
• Side chains project outward.
• Disrupted by proline, glycine, and charged residues.
• Polypeptide chains arranged side-by-side (parallel or antiparallel).
• Stabilized by interchain hydrogen bonds.
• More extended than α-helix.
• Sharp bends connecting helices and sheets.
• Often contain glycine (flexible) or proline (kink-forming).
One region forms a helical coil stabilized by hydrogen bonds; another forms zig-zag strands lying side by side, connected by hydrogen bonds.
The overall 3-dimensional shape of a single polypeptide chain.
• Hydrophobic interactions – bury nonpolar residues inward.
• Hydrogen bonds – stabilize external and internal interactions.
• Ionic bonds (salt bridges) – between charged side chains.
• Disulfide bonds – covalent S–S links between cysteines.
• Van der Waals forces – tight packing in the protein core.
• Independently folded functional units (e.g., catalytic domains, binding domains).
Hydrophobic residues cluster inward while polar and charged residues orient outward, creating a compact, functional 3D structure.
Association of two or more polypeptide chains (subunits) into a functional protein.
• Subunits held together by noncovalent interactions (H-bonds, ionic, hydrophobic)
• Sometimes by interchain disulfide bonds.
• Allows cooperativity, regulation, and structural stability.
• Hemoglobin – α₂β₂ tetramer
• Immunoglobulins – multiple polypeptide chains linked by disulfide bonds
• Lactate dehydrogenase – tetrameric enzyme
Multiple folded subunits assemble into a larger complex, each occupying a precise position and contributing to overall function.
• Human insulin is composed of two polypeptide chains:
• 21 amino acids
• Contains an intra-chain disulfide bond between A6–A11
• N-terminus begins with Glycine, C-terminus ends with Asparagine
• 30 amino acids
• N-terminus begins with Phenylalanine, C-terminus ends with Threonine
Insulin contains three disulfide bridges:
A7 – B7 (inter-chain)
A20 – B19 (inter-chain)
A6 – A11 (intra-chain)
• Insulin is synthesized as preproinsulin → proinsulin → insulin.
• C-peptide connects A- and B-chains in proinsulin and is removed during maturation.
• C-peptide is biologically inactive but a marker of insulin secretion.
• The primary structure (specific amino-acid sequence + disulfide pattern) is essential for insulin’s ability to bind its receptor.
• Correct folding of A- and B-chains requires the precise disulfide pattern.
• Any alteration (mutation, reduction, or improper re-oxidation) → loss of biological activity.
• These bonds hold insulin in its functional conformation, enabling receptor binding.
• Intra-chain disulfide that stabilizes α-helical structure.
• Mutations in A-chain regions that stabilize the helix → rapid degradation or poor receptor binding.
• The N-terminal residues of the B-chain (Phe1, Val2, Asn3) are critical for insulin receptor recognition.
• B-chain C-terminal residues also participate in the interaction with the receptor.
• Mutations in the B-chain reduce potency, binding, and half-life.
• In pancreatic β-cells, insulin forms hexamers with zinc.
• Hexamer → storage form
• Monomer → biologically active form
• Rapid-acting insulin analogs are designed to prevent hexamer formation, improving absorption.
• Removed before secretion.
• Its presence prevents early folding and aggregation during insulin synthesis.
• Used as a clinical marker for endogenous insulin production.
• Point mutations in the insulin gene can disturb folding → “insulinopathies.”
• Examples include improper disulfide pairing or disrupted receptor domains leading to early-onset diabetes.
• Sequence analysis determines the exact order of amino acids in a protein.
• Essential for understanding protein structure, function, and genetic mutations.
1. Edman Degradation
• Sequential removal of N-terminal amino acids one at a time.
• Each residue is identified → gives primary structure.
• Useful for peptides up to ~50 residues.
2. Enzymatic Cleavage
• Proteases like trypsin, chymotrypsin, pepsin cut proteins at specific residues.
• Fragments are analyzed and assembled into the full sequence.
3. Chemical Cleavage
• Cyanogen bromide cleaves at methionine residues.
4. Mass Spectrometry
• Modern method for rapid peptide sequencing.
• Identifies amino acids based on mass/charge ratios.
• Detects post-translational modifications (phosphorylation, acetylation, disulfide patterns).
• Identification of mutations (e.g., sickle cell anemia: Glu→Val).
• Protein engineering & recombinant insulin design.
• Quality control in biotech proteins.
• pI is the pH at which an amino acid or protein has zero net charge.
• Exists predominantly as a zwitterion.
• Minimum solubility at pI → proteins precipitate easily.
• No movement in an electric field → basis of isoelectric focusing.
• Proteins with a high content of acidic residues → low pI.
• Proteins rich in basic residues → high pI.
• For neutral amino acids:
pI = (pK₁ + pK₂) / 2
• For acidic and basic amino acids: all three pKs considered.
• Abnormal pI helps detect protein variants in diseases (e.g., HbS, HbC).
• Used in separation of serum proteins (albumin, globulins).
Proteins precipitate when their solubility decreases, caused by pH, salts, organic solvents, or heat.
• Proteins have minimum solubility at pI.
• Used to isolate casein from milk at pH 4.6.
• Used in laboratory protein purification.
• High salt concentration (ammonium sulfate) removes water from proteins.
• Protein–protein interactions increase → precipitation.
• Common in protein purification and enzyme isolation.
• Alcohols (ethanol, acetone) reduce dielectric constant → protein denaturation → precipitation.
• Used in plasma protein fractionation.
• Hg²⁺, Pb²⁺, Ag⁺ bind –COO⁻ and –SH groups → protein precipitation.
• Used in poisoning treatment (egg white protects gut mucosa).
• Basis of some diagnostic tests.
• High temperature disrupts hydrogen bonds and hydrophobic interactions → coagulation.
• Example: egg white becoming solid on heating.
• Extreme pH denatures proteins.
• Used in gastric digestion (HCl).
• Used to precipitate casein or remove proteins from solutions.
• Mild denaturing agents (urea, guanidinium chloride) disrupt tertiary structure.
• Leads to exposure of hydrophobic groups and aggregation.
• Denaturation is the loss of secondary, tertiary, or quaternary structure of a protein without breaking peptide bonds.
• The primary structure remains intact, but the protein loses shape and function.
• Hydrogen bonds, ionic bonds, hydrophobic interactions, and disulfide bonds are disrupted.
• Protein unfolds → loss of biological activity.
• Hydrophobic groups become exposed → aggregation or precipitation.
• Heat
• Strong acids or alkalis
• Organic solvents (alcohol, acetone)
• Heavy metals (Hg²⁺, Pb²⁺)
• Detergents (SDS)
• Reducing agents (β-mercaptoethanol)
• Radiation or ultrasonic vibration
• Loss of enzymatic activity
• Loss of solubility → precipitation
• Change in physical properties (viscosity, optical rotation)
• Sometimes reversible, but usually irreversible in biological systems
• Cooking an egg (albumin coagulation)
• Denaturation of enzymes during fever
• Alcohol-based hand sanitizers denature viral proteins
• HCl in the stomach denatures dietary proteins
• Essential in digestion, diagnostics, food processing, and understanding protein folding diseases.
• Coagulation is precipitation of proteins due to heat, caused by irreversible denaturation and aggregation.
• Heat breaks weak non-covalent bonds (hydrogen bonds, hydrophobic interactions).
• Protein unfolds → exposed hydrophobic regions stick together → insoluble coagulum forms.
• Egg white (albumin) turning solid when boiled
• Coagulation of milk proteins when heated
• Clotting of serum proteins during diagnostic heat tests
• Heat-labile enzymes losing activity at high temperatures
• Heat coagulation test for detecting proteins in urine or cerebrospinal fluid.
• Fever can partially denature heat-sensitive enzymes, affecting metabolism.
• Heat instability in certain genetic enzyme deficiencies.
• Denaturation = unfolding
• Coagulation = unfolding + aggregation + precipitation
A protein heated above its stability range begins to unfold, exposing hydrophobic inner residues. These residues stick to one another, forming large aggregates that become insoluble and appear as a solid mass or cloudy precipitate.
Proteins can be classified based on composition, shape, and function.
• Yield only amino acids on hydrolysis.
• Examples:
– Albumins (serum albumin)
– Globulins (IgG)
– Histones
• Protein + non-protein (prosthetic) group.
Examples:
• Glycoproteins → carbohydrate
• Lipoproteins → lipids
• Metalloproteins → metal ions (hemoglobin, cytochromes)
• Phosphoproteins → phosphate (casein)
• Nucleoproteins → nucleic acids
• Formed by partial hydrolysis or denaturation.
• Examples: metaproteins, proteoses, peptones, polypeptides.
• Long, thread-like
• Structural role
• Low solubility
• Examples: Collagen, Keratin, Elastin
• Spherical, compact
• Functional role (enzymes, hormones)
• Soluble in water
• Examples: Enzymes, Hemoglobin, Albumin
• Enzymatic (lipase, trypsin)
• Structural (collagen, keratin)
• Transport (hemoglobin)
• Storage (ferritin)
• Regulatory (insulin)
• Protective (antibodies)
Common biochemical methods used to measure protein concentration:
• Based on violet complex formed between Cu²⁺ and peptide bonds in alkaline medium.
• Requires two or more peptide bonds → minimum tripeptide.
• Used for plasma proteins.
• More sensitive than Biuret.
• Combines Biuret reaction + reduction of Folin–Ciocalteu reagent → blue color.
• Sensitive to aromatic amino acids.
• Uses Coomassie Brilliant Blue dye.
• Dye binds to basic and aromatic residues → blue color.
• Very sensitive, widely used for micro-assays.
• Aromatic residues (Phe, Tyr, Trp) absorb UV at 280 nm.
• Rapid method for pure proteins.
• Measures total nitrogen content; indirectly measures total protein.
• Used in food analysis.
• Folding converts a linear polypeptide into its functional 3D conformation.
• Driven by:
– Hydrophobic interactions
– Hydrogen bonds
– Ionic interactions
– Disulfide bonds
– Van der Waals forces
• Folding follows a path toward a stable low-energy state (native structure).
• Incorrect folding → aggregation → diseases (Alzheimer’s, Parkinson’s).
• Specialized proteins that assist in proper folding without becoming part of the final protein.
• Hsp60 (Chaperonins) — barrel-shaped; provide isolated environment for folding.
• Hsp70 — binds hydrophobic regions and prevents aggregation.
• Protein disulfide isomerase (PDI) — rearranges disulfide bonds.
• Peptidyl-prolyl isomerase — converts cis/trans proline bonds.
• Prevent misfolding
• Promote refolding
• Assist in transport across membranes
• Prevent aggregation during stress
• Some denatured proteins can regain their native structure and function when denaturing agents are removed.
• Works only if the primary structure is intact.
• Demonstrates that all information for proper folding lies in the amino-acid sequence.
• Ribonuclease regains full activity after denaturation by urea and β-mercaptoethanol when reagents are removed.
• Not all proteins renature easily.
• Aggregated proteins generally cannot renature.
• In cells, chaperones are required to guide renaturation.
Proteins perform diverse biological roles essential for life.
• Proteins act as biocatalysts (enzymes).
• Increase reaction rate without being consumed.
• Examples: amylase, lipase, DNA polymerase.
• Provide strength, shape, and support.
• Examples: collagen, keratin, elastin, actin, tubulin.
• Carry molecules across membranes or in blood.
• Examples: hemoglobin (O₂), transferrin (iron), albumin (fatty acids & drugs).
• Hormonal and signaling proteins regulate metabolism.
• Examples: insulin, glucagon, growth hormone.
• Immunoglobulins defend against infection.
• Complement proteins help destroy pathogens.
• Fibrinogen involved in clot formation.
• Ferritin stores iron.
• Casein stores amino acids in milk.
• Actin and myosin enable muscle contraction.
• Dynein and kinesin move organelles inside cells.
• Proteins help maintain acid–base balance due to amphoteric nature.
• Membrane-bound receptors bind hormones, neurotransmitters, and antigens.
Plasma proteins include albumin, globulins, and fibrinogen.
• Major plasma protein (~60%).
• Maintains oncotic pressure → prevents edema.
• Transports fatty acids, bilirubin, calcium, drugs.
• Reduced in malnutrition, liver disease, nephrotic syndrome.
• α and β globulins
– Transport proteins (transferrin, ceruloplasmin, lipoproteins).
• γ-globulins (Immunoglobulins)
– Antibodies: IgG, IgA, IgM, IgE, IgD.
• Precursor of fibrin for blood clot formation.
• Synthesized in liver.
• Normal: 1.2–1.8.
• Low ratio → increased globulins (infection), low albumin (liver disease).
• Used in diagnosing liver disease, kidney disease, malnutrition, inflammation.
• Electrophoresis helps detect multiple myeloma.
• Most abundant protein in the body.
• Triple-helical structure: three polypeptide chains wound into a rope-like helix.
• Rich in glycine, proline, and hydroxyproline.
• Type I – bone, skin, tendon
• Type II – cartilage
• Type III – blood vessels
• Type IV – basement membrane (network-forming)
• Requires vitamin C for hydroxylation of proline and lysine.
• Cross-linking via lysyl oxidase requires copper.
• Scurvy (impaired hydroxylation)
• Osteogenesis imperfecta (defective Type I collagen)
• Ehlers–Danlos syndrome (defective cross-linking)
• Highly elastic protein allowing tissues to stretch and recoil.
• Rich in glycine, alanine, valine.
• Contains unique amino acids desmosine and isodesmosine → cross-links.
• Found in ligaments, lungs, skin, arterial walls.
• Random coil structure → elasticity.
• Cross-linking provides resilience and recoil.
• Marfan syndrome — defective fibrillin (scaffolding protein for elastin).
• Leads to hyperelastic joints, lens dislocation, aortic aneurysm.
It is the linear sequence of amino acids linked by peptide bonds.
Because it has partial double-bond character due to resonance, restricting rotation.
Hydrogen bonds between the carbonyl oxygen and amide hydrogen of the peptide backbone.
A right-handed helical structure with 3.6 residues per turn, stabilized by intrachain hydrogen bonds.
Proline, glycine, and clusters of charged residues.
A stretched peptide arrangement stabilized by interchain hydrogen bonds; can be parallel or antiparallel.
Hydrophobic interactions, hydrogen bonds, ionic bonds, van der Waals forces, and disulfide bonds.
A compact, independently folded, functional region within a protein.
Association of two or more polypeptide subunits into a functional protein.
Hemoglobin, immunoglobulins, LDH (tetramer).
Proteins that yield only amino acids on hydrolysis (e.g., albumin, globulin).
Proteins with a prosthetic group (e.g., hemoglobin, lipoproteins, glycoproteins).
Maintains oncotic pressure and transports fatty acids, bilirubin, and drugs.
Albumin–globulin ratio; normal is 1.2–1.8.
Glycine, proline, and hydroxyproline.
Required for hydroxylation of proline and lysine.
Impaired hydroxylation → unstable collagen.
Contains desmosine and isodesmosine, which provide elasticity.
Defective fibrillin, which is required for elastin scaffolding.
Denaturation and precipitation of proteins on heating (e.g., cooking an egg).
Biuret method (violet complex with peptide bonds).
Binding of Coomassie Blue dye to basic and aromatic residues.
Recovery of native protein structure after removal of denaturing agents—possible only if the primary structure remains intact.
Chaperones, including Hsp70, Hsp60 (chaperonins), and PDI.
It may aggregate and cause diseases like Alzheimer’s, Parkinson’s, and prion diseases.
A. Ionic bonding
B. Hydrogen bonding
C. Resonance causing partial double-bond character
D. Hydrophobic interactions
Answer: C
A. Alanine
B. Proline
C. Valine
D. Cysteine
Answer: B
A. Disulfide bonds
B. Hydrogen bonds between different segments
C. Hydrophobic interactions
D. Van der Waals forces
Answer: B
A. Single chain of myoglobin
B. Triple helix of collagen
C. α₂β₂ arrangement of hemoglobin
D. α-helix in keratin
Answer: C
A. Globulin
B. Fibrinogen
C. Albumin
D. Transferrin
Answer: C
A. IgG
B. Albumin
C. Fibrinogen
D. Ceruloplasmin
Answer: B
A. Elastin
B. Keratin
C. Collagen
D. Albumin
Answer: C
A. Glycosylation
B. Hydroxylation
C. Disulfide bond formation
D. Cleavage of propeptides
Answer: B
A. Hydroxylysine
B. Desmosine and isodesmosine
C. Selenocysteine
D. Ornithine
Answer: B
A. Scurvy
B. Marfan syndrome
C. Osteogenesis imperfecta
D. Ehlers–Danlos syndrome
Answer: B
A. Reaction with aromatic rings
B. Copper binding to peptide bonds
C. Reaction with sulfhydryl groups
D. Reduction of Folin reagent
Answer: B
A. Ninhydrin binding
B. Protein absorption at 280 nm
C. Coomassie Blue dye binding to proteins
D. Reduction of metal ions
Answer: C
A. Peptide bond formation
B. Proper protein folding
C. DNA replication
D. Protein degradation
Answer: B
A. Disulfide bonds break
B. Primary structure remains intact
C. Protein is heat-coagulated
D. Aggregates are present
Answer: B
A. Hemoglobin
B. Lipoprotein
C. Albumin
D. Glycoprotein
Answer: C
A. One
B. Two
C. Three
D. Four
Answer: C
A. Plasma albumin
B. Immunoglobulins
C. Fibrinogen
D. Transferrin
Answer: B
A. Peptide bond hydrolysis
B. Breaking of weak non-covalent interactions
C. Increased hydrogen bonding
D. Enhanced tertiary folding
Answer: B
A. Hsp70
B. PDI (Protein disulfide isomerase)
C. Peptidyl-prolyl isomerase
D. Trypsin
Answer: B
A. Albumin
B. Fibrinogen
C. γ-Globulins
D. α-Globulins only
Answer: C
The linear sequence of amino acids linked by peptide bonds.
Because resonance gives the –CO–NH– bond partial double-bond character, restricting rotation.
Hydrogen bonds between backbone –C=O and –NH groups.
A right-handed helix stabilized by intrachain hydrogen bonds; 3.6 residues per turn.
Proline (kink-forming) and glycine (too flexible).
Extended strands arranged side-by-side, stabilized by interchain hydrogen bonds.
Hydrophobic interactions, hydrogen bonds, ionic bonds, van der Waals forces, and disulfide bonds.
A compact, independently folded functional region.
Association of two or more polypeptide chains into a functional protein.
Hemoglobin (α₂β₂).
Maintains plasma oncotic pressure and transports fatty acids, bilirubin, and drugs.
The albumin–globulin ratio (normal 1.2–1.8).
Glycine, proline, hydroxyproline.
Required for hydroxylation of proline and lysine residues.
Impaired hydroxylation → unstable collagen.
Cross-linking via desmosine and isodesmosine.
Fibrillin, the scaffolding protein for elastin.
Disruption of weak non-covalent bonds by heat → unfolding + aggregation.
Copper ions bind to peptide bonds in alkaline medium → violet color.
A denatured protein regains its native structure when the denaturant is removed, provided the primary structure is intact.
Assist correct protein folding and prevent aggregation.
Protein disulfide isomerase (PDI).
A protein that yields only amino acids on hydrolysis (e.g., albumin).
Protein + prosthetic group (e.g., hemoglobin, lipoprotein, glycoprotein).
They are amphoteric, containing both acidic and basic groups.
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