MCBG Session 10 - Protein Structure

Question 1

Outline the binding of oxygen to haemoglobin.

Answer 1

• O₂ binds to myoglobin and haemoglobin via the Fe atom in the haem prosthetic group.
• O₂ binds to myoglobin in a hyperbolic manner dependent on oxygen concentration.
• Adult haemoglobin has 4 haem-containing subunits, ₂ a and ₂ b, each of which can bind a molecule of O₂
• Haemoglobin can exist in 2 different interchangeable states, a low affinity T state and a high affinity R state.
• Binding of oxygen to one subunit of haemoglobin causes conformational changes which promotes the R state.
• The sigmoidal binding curve for oxygen binding to haemoglobin is physiologically important as it allows for efficient oxygen binding and delivery.
• Oxygen binding to haemoglobin is dependent on CO₂ , H+ and BPG concentration. These effectors work by changing stabilising the T state.
• Oxygen binding to haemoglobin can be disrupted by inhibitors (CO) or genetic disease (sickle cell anaemia)
• Appreciate that mutations in globin genes can give rise to diseases such as thalassemia.

Question 2

Outline the structure, function and importance of myoglobin.

Answer 2

• Myoglobin and haemoglobin are haem proteins whose physiological importance is principally related to their ability to bind molecular oxygen.
• Myoglobin is a monomeric haem protein found mainly in muscle tissue where it serves as an intracellular storage site for oxygen. During periods of oxygen deprivation oxymyoglobin releases its bound oxygen which is then used for metabolic purposes.
• Myoglobin is the primary oxygen-carrying pigment of muscle tissues.
• High concentrations of myoglobin in muscle cells allow organisms to hold their breath for a longer period of time.

Question 3

What is the structure, function and importance of haemoglobin?

Answer 3

• Myoglobin and haemoglobin are haem proteins whose physiological importance is principally related to their ability to bind molecular oxygen.
• Haemoglobin in the blood carries oxygen from the respiratory organs (lungs or gills) to the rest of the body (i.e. the tissues).
• There it releases the oxygen to permit aerobic respiration to provide energy to power the functions of the organism in the process called metabolism

Question 4

In detail, outline oxygen binding to haemoglobin and myoglobin.

Answer 4

• Haem consists of a protoporphyrin ring and an Fe atom bound to 4 N atoms of the ring.
• Fe²⁺ can make 2 additional bonds to oxygen, one on either side of the plane.
• 1 molecule of O₂ binds to the haem group in myoglobin and haemoglobin.
• Fe atom is bound to the protein via a histidine residue (proximal histidine) on the other side of the ring.

Question 5

Explain oxygen binding to haem in myoglobin.

Answer 5

• Fe in deoxymyoglobin is slightly below the plane of the ring.
• Oxygen binding causes the movement of Fe into the plane of the ring
• Movement causes movement of His F8 and small change in the overall protein conformation.
• Oxygen binding to myoglobin shows a hyperbolic dependence on oxygen concentration.

Question 6

Compare the different oxygen dissociation curves for myoglobin and haemoglobin.

Answer 6

Question 7

Haemoglobin undergoes a structural change when binding to oxygen. Explain this

Answer 7

• Deoxyhaemoglobin can exist in low affinity T state or high affinity R state
• Oxygen binding promotes stabilisation of the R state.

Question 8

Outline the co-operative binding of oxygen.

Answer 8

• Binding affinity for oxygen increases as more oxygen molecules bind to Hb subunits
• Binding of 1st O_² molecule to 1 subunit is hard – low affinit

- Binding of last O₂ molecule to 4th subunit is very easy – high affinity

Question 9

Outline ‘co-operative binding’ of oxygen to haemoglobin.

Answer 9

• The binding of one oxygen molecule promotes the binding of subsequent molecules.
• The sigmoidal binding curve of haemoglobin means that O₂ can be efficiently carried from the lungs to the tissues
• More sensitive to small difference in O₂ concentrations,

Question 10

Outline the regulation of oxygen binding in terms of 2,3-Bisphosphoglycerate (BPG)

Answer 10

• 1 BPG binds per haemoglobin tetramer and decreases the affinity for O₂.
• BPG concentration increases at high altitudes, promoting O₂ release at the tissues.

Question 11

Outline the regulation of oxygen binding in terms of CO₂ and H⁺

Answer 11

• H⁺ and CO₂ can both bind to haemoglobin molecules.
• Binding of H⁺ and CO₂ lowers the affinity of haemoglobin for oxygen.

Question 12

Outline the regulation of oxygen binding in terms of Carbon monoxide poisoning.

Answer 12

• Carbon monoxide (CO) is a poison because it combines with ferromyoglobin and ferrohaemoglobin and blocks oxygen transport.
• CO binds to haemoglobin 250x more readily than O₂

Question 13

Explain the importance of haemoglobin.

Answer 13

• HbF is the major haemoglobin in foetal blood.
• Higher binding affinity for O₂ than HbA which allows transfer of O₂ to foetal blood supply from the mother.

Question 14

Outline the features of sickle cell anaemia.

Answer 14

• Mutation of glutamate to Valine in β globin (HbS)
• “Sticky” hydrophobic pocket formed by Val allows deoxygenated HbS to polymerise.
• Sickled cells are:

I. More prone to lyse (anaemia)

II, More rigid (black microvasculature)

Question 15

Outline the features of thalassemia.

Answer 15

• Thalassemia: are a group of genetic disorders where there is an imbalance between the number of α- and β-globin chains
• β-thalassaemias

I. Decreased or absent β-globin chain production

II. α-chains unable to form stable tetramers

III. Symptoms appear after birth

• α-thalassaemias

I. Decreased or absent α-globin chain production

II. Several different levels of severity due to the multiple copies of the α-chains present

• β-chains can form stable tetramers with increased affinity for oxygen
• Onset before birth

Question 16

Proteins may be delivered to the plasma membrane via the regulated or secretory constitutive pathway

Answer 16

• Constitutive secretory pathway:

I. Immunoglobulins

II. Albumin

III. Prohormones

IV. Collagen

V. Receptors

• Regulated secretory pathway:

I. Insulin

II. Glucagon

III. Trypsin

Question 17

What is collagen?

Answer 17

• Collagen is a secreted protein produced by fibroblasts in the connective tissue.
• It is the most abundant protein in the body (25-35%).
• It is also the most abundant fibrous protein in connective tissue.
• It composes the tendons, ligaments, cartilage and bone. It also composes the loose connective tissue, providing structure to internal organs.

Question 18

Outline the structure of collagen.

Answer 18

• Basic unit is Tropocollagen:
• 3 polypeptides (a chains), each ~1000aa long
• 300nm rod-shaped protein
• Glycine in every 3^rd position along each a chain i.e. (Gly-X-Y)_n repeat
• Characteristic triple helix (right-handed)
• Mostly proline or hydroxyproline in X and some Y positions
• H-bonds between a chains stabilise structure

Question 19

Outline the triple helix structure of collagen.

Answer 19

Triple helix structure:

• Non-extensible
• Non-compressible
• High tensile strength
• Glycine is the only amino acid with a side chain small enough to fit in the middle of the helix

Question 20

Outline the steps involved in the synthesis and modification of collagen in the ER.

Answer 20

1. Synthesis and entry of chain into lumen of rough ER
2. Cleavage of signal peptide (Prepro α chains → pro α chains)
3. Hydroxylation of selected proline and lysine residues
4. Addition of N-linked oligosaccharides
5. Addition of galactose to hydroxylysine residues
6. Chain alignment, formation of disulphide bonds.
7. Formation of triply-helical procollagen from C- to N-terminus
8. Completion of O-linked oligosaccharide chains by addition of glucose
9. Transport vesicle
10. Exocytosis
11. Removal of N- and C-terminal propeptides
12. Lateral association of collagen molecules followed by covalent cross-linking
13. Aggregation of fibrils

Question 21

What is prolyl hydroxylase?

Answer 21

• Requires vitamin C and Fe⁺² ions for activity
• Associated with PDI in the ER
• Allows increased H-bonding to stabilise triple helix
• Scurvy is due to weak tropocollagen triple helices