Hemoglobin Cooperativity, Structure, and Function in Oxygen Transport

What causes positive cooperativity in hemoglobin?

The binding of O₂ to one subunit stabilizes the R state, increasing the affinity of remaining subunits for O₂.

What causes negative cooperativity in a protein?

Binding of one ligand decreases the affinity at other sites by stabilizing a conformation less favorable for additional binding.

What structural feature allows hemoglobin to exhibit cooperativity?

Its quaternary structure of four subunits connected by interfaces that transmit conformational changes.

What stabilizes the T (tense) state of hemoglobin?

Salt bridges and hydrogen bonds between subunits that lower oxygen affinity.

What stabilizes the R (relaxed) state of hemoglobin?

Oxygen binding that breaks salt bridges, pulls Fe²⁺ into the porphyrin plane, and increases affinity.

What happens when O₂ binds to one subunit of hemoglobin?

The Fe²⁺ moves into the heme plane, the His side chain shifts, and subunit rotation triggers the T → R transition.

How does 2,3-BPG regulate hemoglobin function?

2,3-BPG binds in the positively charged central cavity of deoxyhemoglobin, stabilizing the T state and lowering oxygen affinity.

What is the physiological effect of 2,3-BPG binding?

It shifts the oxygen binding curve to the right, promoting oxygen release in tissues.

How does altitude adaptation involve 2,3-BPG?

At high altitude, 2,3-BPG levels rise to favor oxygen unloading in tissues despite lower atmospheric O₂.

Why does fetal hemoglobin bind oxygen more tightly than adult hemoglobin?

Fetal Hb has γ-chains instead of β-chains, which reduce 2,3-BPG binding, favoring the R state and higher O₂ affinity.

What is the Bohr effect?

The inverse relationship between pH and oxygen affinity: low pH stabilizes the T state and promotes O₂ release.

Mechanistically, how does the Bohr effect occur?

Protonation of His146 at low pH forms a salt bridge with Asp94, stabilizing the T state and decreasing O₂ affinity.

What happens to hemoglobin's O₂ curve at low pH?

It shifts to the right — indicating decreased affinity and enhanced O₂ delivery.

What happens to hemoglobin's O₂ curve at high pH?

It shifts to the left — increased affinity and tighter O₂ binding.

How does CO₂ affect hemoglobin function?

CO₂ reacts with N-terminal amino groups to form carbamates, releasing H⁺ and stabilizing the T state to promote O₂ release.

Why is carbon monoxide (CO) toxic?

It binds Fe²⁺ in heme about 250× more tightly than O₂, locking hemoglobin in the R state and preventing O₂ release.

Why is free Fe²⁺ dangerous for oxygen transport?

It oxidizes to Fe³⁺, generating reactive species and losing oxygen-binding capacity.

What structural change occurs in heme upon O₂ binding?

Fe²⁺ is pulled into the porphyrin plane, reducing the dome shape of the heme and initiating the T → R transition.

What amino acid substitution causes sickle-cell anemia?

Glu6 → Val in the β-chain of hemoglobin.

How does this mutation alter hemoglobin's properties?

The nonpolar valine creates a hydrophobic patch on the β-chain, causing HbS to polymerize in the deoxy form.

What is the physiological consequence of hemoglobin polymerization in sickle-cell disease?

Red blood cells become rigid and sickle-shaped, blocking capillaries and reducing oxygen delivery.

What forces drive protein folding?

The hydrophobic effect, hydrogen bonding, van der Waals interactions, and electrostatic attractions.

Why does the hydrophobic effect drive folding?

Nonpolar side chains cluster inside the protein, releasing ordered water molecules and increasing overall entropy.

What stabilizes tertiary protein structure?

Hydrophobic core packing, hydrogen bonds, salt bridges, and disulfide bonds.

What role do disulfide bonds play in protein stability?

They covalently link cysteine residues, locking the protein into a stable conformation.

What did the Anfinsen experiment prove?

A protein's native structure is determined entirely by its amino acid sequence.

What do chaperone proteins do?

They prevent aggregation of unfolded proteins and assist in proper folding by shielding hydrophobic regions.

How do chaperonins like GroEL/ES assist folding?

They encapsulate misfolded proteins in an ATP-dependent chamber where refolding occurs without aggregation.

What is the common feature of proteins that misfold?

They expose hydrophobic residues that promote aggregation into insoluble fibrils or plaques.

Why is protein misfolding dangerous?

It leads to aggregation that can damage cells, as seen in Alzheimer's or prion diseases.

What gives the peptide bond partial double-bond character?

Resonance between the carbonyl oxygen and amide nitrogen.

What effect does this have on peptide bond rotation?

It restricts rotation around the C-N bond, making the peptide bond planar and rigid.

What are φ (phi) and ψ (psi) angles?

Angles of rotation around the N-Cα and Cα-C bonds that define backbone conformation.

What does a Ramachandran plot show?

The allowed combinations of φ and ψ angles that correspond to stable secondary structures.

What stabilizes an α-helix?

Hydrogen bonds between the backbone N-H and C=O groups of residues four apart (n and n+4).

Why do proline and glycine disrupt α-helices?

Proline's rigid ring breaks hydrogen bonding; glycine's flexibility destabilizes the helix.

What stabilizes β-sheets?

Hydrogen bonds between the backbone amides and carbonyls of adjacent strands.

Why are antiparallel β-sheets stronger than parallel ones?

Their hydrogen bonds are linear, providing optimal strength and alignment.

What amino acids are common in β-turns?

Proline (position 2) and glycine (position 3) because of their geometry and flexibility.

What catalyzes cis-trans isomerization of proline bonds?

Proline isomerase enzymes.

What distinguishes fibrous from globular proteins?

Fibrous proteins provide structure and are water-insoluble; globular proteins are compact, dynamic, and soluble.

Describe α-keratin's structure.

Two right-handed α-helices coil into a left-handed supercoil, stabilized by hydrophobic interactions and disulfide bonds.

How does a permanent wave in hair work?

Disulfide bonds in keratin are reduced, reshaped, and reoxidized to set a new configuration.

Describe collagen's triple-helix structure.

Three left-handed helices twist into a right-handed supercoil rich in glycine and hydroxyproline.

What stabilizes collagen's triple helix?

Hydrogen bonding and tight packing of glycine at the core of the triple helix.

Why is vitamin C necessary for collagen formation?

It keeps Fe²⁺ in prolyl hydroxylase reduced, enabling hydroxylation of proline to hydroxyproline.

What happens to collagen when vitamin C is deficient?

Hydroxylation fails, weakening hydrogen bonding and causing fragile connective tissue (scurvy).

Describe silk fibroin structure.

Antiparallel β-sheets packed closely due to small side chains (Gly, Ala) stabilized by H-bonds and dispersion forces.

Why is spider silk both strong and elastic?

It combines crystalline β-sheet regions for strength and amorphous regions for stretch.

What drives tertiary structure formation?

The burying of hydrophobic side chains and formation of stabilizing intramolecular interactions.

What are motifs in proteins?

Recurrent combinations of α-helices and β-sheets that form recognizable structural patterns (e.g., β-α-β loop).

What are intrinsically disordered proteins?

Proteins or regions lacking fixed structure that can adapt to multiple partners, often in signaling.

How does X-ray crystallography determine structure?

It measures diffraction patterns from crystallized proteins to model atomic positions.

How does NMR spectroscopy determine structure?

It detects magnetic interactions of atoms in proteins in solution to determine structure and dynamics.

What is the functional role of myosin?

Myosin uses ATP hydrolysis to generate conformational changes that move along actin filaments for contraction.

How does ATP regulate myosin's interaction with actin?

ATP binding releases myosin from actin; hydrolysis re-cocks the head for the next power stroke.

How do troponin and tropomyosin control contraction?

Tropomyosin blocks actin sites until Ca²⁺ binds to troponin, shifting tropomyosin to expose binding sites.

What triggers muscle contraction?

A nerve impulse releases Ca²⁺ from the sarcoplasmic reticulum, allowing actin-myosin interaction.

How do antibodies recognize antigens?

Through variable regions forming complementary shapes and charge distributions with specific epitopes.

What type of binding occurs between antigen and antibody?

Noncovalent induced-fit interactions including hydrogen bonds, ionic, and hydrophobic forces.

What is an epitope?

The specific region on an antigen that an antibody binds.

What is the structure of IgG?

Two heavy and two light chains linked by disulfide bonds; variable regions form two antigen-binding sites.

What differentiates cellular and humoral immunity?

Cellular immunity uses T cells and macrophages to kill infected cells; humoral immunity uses antibodies to neutralize extracellular pathogens.