BS1030 Topic 2 Lecture 1 Tertiary and quaternary structure and function of haemoglobin

What are the four levels of protein structure?

Primary - amino acid sequence; Secondary - α-helices and β-sheets; Tertiary - 3D folding of one chain; Quaternary - assembly of multiple subunits.

What is tertiary structure?

The overall 3D folding of a single polypeptide chain, composed of secondary structure elements linked by loops and turns.

What stabilises protein tertiary and quaternary structures?

Noncovalent bonds (hydrogen bonds, ionic bonds, van der Waals forces, hydrophobic interactions) and covalent disulphide bridges.

What are the noncovalent forces stabilising protein folding?

Hydrogen bonds, ionic bonds (salt bridges), van der Waals forces, and the hydrophobic effect.

What are covalent bonds that stabilise some proteins?

Disulphide (S-S) bridges formed between cysteine residues.

Where are disulphide bridges commonly found?

In proteins that function in harsh or extracellular environments (e.g., ribonuclease A).

What is the hydrophobic effect?

The tendency for hydrophobic side chains to cluster inside the protein, away from water, increasing water entropy and driving folding.

What is the thermodynamic equation for protein folding?

ΔG = ΔH - TΔS.

What does ΔG < 0 mean for protein folding?

Folding is thermodynamically favourable (spontaneous).

Why is ΔG of folding relatively small (~-40 kJ/mol)?

Because folding is easily disrupted by temperature, pH, or mutations.

What did the Anfinsen experiment demonstrate?

That the amino acid sequence contains all the information required for a protein to fold into its correct 3D structure.

Who conducted the Anfinsen experiment and when?

Christian Anfinsen, 1963.

What protein did Anfinsen study?

Ribonuclease A.

What is quaternary structure?

The overall 3D arrangement of multiple folded subunits in a functional protein (e.g., haemoglobin α₂β₂).

What are post-translational modifications (PTMs)?

Chemical modifications made to proteins after translation that regulate their function or stability.

Give examples of common post-translational modifications.

Phosphorylation, methylation, acetylation, glycosylation, and ubiquitination.

What are the effects of post-translational modifications?

They can activate or deactivate enzymes, alter localization, or change stability.

What enzyme catalyses phosphorylation?

A kinase.

What amino acids are typically phosphorylated?

Serine, threonine, or tyrosine residues (on their -OH groups).

What is myoglobin?

A single-chain, single-haem protein found in muscle, responsible for short-term oxygen storage.

What is haemoglobin?

A tetrameric protein (α₂β₂) in red blood cells that transports oxygen from lungs to tissues.

How many haem groups are in haemoglobin?

Four haem groups, one per subunit, allowing binding of four O₂ molecules.

What is the role of the haem group?

It binds oxygen via the iron (Fe²⁺) ion, which is coordinated to a histidine residue in the polypeptide chain.

What structural change occurs upon oxygen binding?

Oxygen binding pulls the iron atom into the haem plane, tugging on the histidine and altering the protein's conformation.

What shape is the O₂-binding curve for myoglobin?

Hyperbolic - constant high affinity for oxygen.

What shape is the O₂-binding curve for haemoglobin?

Sigmoidal - shows cooperative binding.

What does cooperativity mean in haemoglobin?

Binding of one oxygen molecule increases the affinity of remaining subunits for oxygen.

What are the two states of haemoglobin?

T (tense) state - low O₂ affinity; R (relaxed) state - high O₂ affinity.

What type of protein is haemoglobin?

An allosteric protein.

What are allosteric proteins?

Usually multi-subunit proteins that change conformation upon ligand binding or regulator interaction.

What are allosteric regulators?

Molecules that bind to a protein and modify its activity by stabilising either the active or inactive conformation.

What is a positive allosteric regulator?

A molecule that stabilises the R state, increasing oxygen affinity.

What is a negative allosteric regulator?

A molecule that stabilises the T state, decreasing oxygen affinity.

Give an example of a negative allosteric regulator of haemoglobin.

2,3-bisphosphoglycerate (BPG), CO₂, or H⁺ (Bohr effect).

How does 2,3-bisphosphoglycerate (BPG) affect haemoglobin?

It binds to deoxygenated Hb, lowering oxygen affinity and stabilising the T state.

How do CO₂ and H⁺ affect haemoglobin?

They decrease O₂ affinity (Bohr effect), promoting O₂ release in metabolically active tissues.

Why is the Bohr effect physiologically important?

It ensures oxygen is released where CO₂ and acid levels are high (active tissues).

What mutation causes sickle cell disease?

A substitution of glutamate (hydrophilic) with valine (hydrophobic) in the β-globin chain (E6V mutation).

How does the E6V mutation affect haemoglobin?

Introduces a hydrophobic patch on the surface that causes HbS molecules to stick together in the T state.

What structural effect does sickle cell mutation have?

Haemoglobin molecules form long, rigid fibres, distorting red blood cells into a sickle shape.

What are the consequences of sickled red blood cells?

They are fragile (causing anaemia) and rigid (blocking small blood vessels).

Name two scientists known for advancing sickle cell research.

Dr Clarice Reid (USA) and Dr Julie Makani (Tanzania).

How does fetal haemoglobin differ from adult haemoglobin?

Fetal Hb (α₂γ₂) has higher oxygen affinity, enabling oxygen transfer from maternal to fetal blood.

What adaptation allows fetal Hb to have higher O₂ affinity?

It binds BPG less tightly than adult Hb.

What is meant by post translational modification?

Covalent attachment of a protein to a chemical group, sugar and lipid. ATP binds to kinase and kinase takes OH- and adds to amino acid. Phosphorylation can alter its localizations, stabiltiy chnage and conformation.

Why can only Serine, Threonine and Tyrosine be phosphorylated?

This is because they have an OH group on their side chains and are referred to as ' phosphoserine', It is a reversible reaction and phosphates can remove to phosphate and kinase can add the phosphate group.

Example of protein phosphorlyation.

Phosphorylation of a nucleus when a cell goes into mitosis and a cell will organize itself. Lamins will repel each other casing the filament to break down and network disassembly. Loss of lamina leads to a nuclear envelop breakdown.

Ubiquitin

9KDA protein and covalently attached with its COOH group to the proteins NH2. Catalysed by E3 ubiquitin ligase and allows chains of it to be built. It is a reversible reaction and to remove ubiquitin with a DUB enzyme.

Consequences of ubiquitlation

Can cause a protein to be degraded.
- Useful in DNA repair ' polyubiquitation'

How does ubiquitylation of cyclins lead to degradation during cell cycle progression

Causing degradation. The cyclin is important proteins and as its ubiquitinated and the signal tags protein to enter a process which are being degraded and it is a on going process where proteins are constantly being degraded