Describe the general structure and role of PDZ domains.
PDZ domains are common structural motifs (~80–90 AAs) found in signalling proteins across bacteria, yeast, plants, viruses, insects, and vertebrates. They bind to the C-terminal or internal sequences of target proteins, usually near plasma membranes, and play critical roles in assembling signal transduction complexes.
Explain the structural features of PDZ domains.
Each PDZ domain consists of six β-strands (βA–βF) and two α-helices (A and B), arranged in a compact globular fold. Ligand peptides bind in an elongated groove where their β-strand aligns antiparallel to the PDZ βB strand. Binding involves a carboxylate-binding loop between βA and βB, recognising the terminal carboxylate group of the peptide ligand.
How do PDZ domains recognise and bind their target sequences?
PDZ domains bind to the C-terminal four/five residues of target proteins, typically transmembrane receptors or ion channels (high affinity). The consensus sequence contains a hydrophobic residue (Val or Ile) at the C-terminus, while positions –2 and –3 determine specificity. PDZ domains may heterodimerise with other PDZ domains, facilitating regulatory IC signalling. Some PDZ domains (sytenin, CASK, Tiam1, FAP) also bind PIP₂, linking membrane lipids to protein scaffolding.
Give examples of PDZ domain–containing proteins and their binding partners.
PSD-95 (Post-Synaptic Density Protein 95) binds:
NMDA receptor B subunit via PDZ1/PDZ2 (binding motif: –IESDV–COOH).
Kv1.4 K⁺ channel via PDZ1/PDZ2 (motif: –VETDV–COOH).
Neuronal nitric oxide synthase (nNOS) via PDZ2-PDZ interaction.
These examples illustrate PDZ-mediated scaffolding in neuronal synapses.
State the main biological functions of PDZ domain proteins.
Maintaining epithelial cell polarity and morphology.
Organising the postsynaptic density in neurons.
Regulating trafficking and activity of membrane proteins.
Define quaternary structure of proteins and outline its key features.
Quaternary structure refers to the association of two or more polypeptide chains (subunits) into a single, functional complex. Each subunit has its own primary, secondary, and tertiary structure, but together they form an oligomeric protein. Subunits can be identical (homomeric) or different (heteromeric) and are held by non-covalent interactions (hydrogen bonds, hydrophobic interactions, ionic bonds, van der Waals forces) and sometimes disulfide bridges. Proper subunit arrangement is essential for biological activity.
Explain why quaternary structure is important for protein function.
The spatial arrangement of subunits determines cooperative and allosteric behaviour (e.g., haemoglobin’s oxygen binding). Structural alterations in subunit interfaces can drastically change activity. Many metabolic and signalling enzymes require conformational flexibility between subunits for regulation.
Give examples of quaternary protein types and their oligomeric states.
Dimers: Alcohol dehydrogenase (ADH), malate dehydrogenase (MDH).
Tetramers: Lactate dehydrogenase (LDH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), haemoglobin (Hb).
Human LDH exists in five isoenzyme variants due to combinations of A and B subunits.
Describe the forces stabilising quaternary structure.
Hydrogen bonds – stabilise between polar residues.
Hydrophobic interactions – drive nonpolar side chains into the interface core.
Ionic interactions – contribute to charge complementarity.
Van der Waals forces – enhance tight packing.
Disulfide bridges – covalently link subunits, increasing structural stability.
How is quaternary structure experimentally determined?
A variety of analytical and computational methods are used:
Analytical Ultracentrifugation: Determines mass via sedimentation velocity.
Size-Exclusion Chromatography: Estimates molecular weight and subunit association.
FRET (Fluorescence Resonance Energy Transfer): Detects subunit proximity and conformational changes.
X-Ray Crystallography: Provides atomic-resolution 3D structures.
NMR Spectroscopy: Infers size via rotational correlation coefficients.
Dynamic Light Scattering (DLS): Measures hydrodynamic radii of complexes.
Bioinformatics / AI-based prediction: Programs such as AlphaFold-Multimer predict subunit interfaces computationally.
What are the key structural and functional features of oligomeric proteins?
Quaternary structures form spontaneously and rapidly once subunits contact.
The oligomeric form is more stable in aqueous environments than separate subunits.
Subunits may undergo conformational or reorientational changes (basis for allostery).
Quaternary structure is essential in cell signalling, e.g. G-protein coupled receptor (GPCR) systems involving α, β, and γ subunits.
Discuss the role of quaternary structure in G-protein–coupled signalling.
GPCRs interact with heterotrimeric G-proteins composed of α, β, and γ subunits. Upon activation, the G-protein binds the receptor, triggering conformational change and downstream signalling. This exemplifies how quaternary association underlies molecular communication and regulation.
Describe the quaternary structure of haemoglobin and its functional significance.
Haemoglobin (Hb) is a tetramer consisting of two α and two β subunits.
Each subunit contains a heme group composed of a ferrous ion (Fe²⁺) coordinated by a porphyrin ring.
The α- and β-chains each contain 7–8 α-helices (A–H), forming a compact globular fold.
Hb exists in two main conformations:
T (tense) state: Low oxygen affinity (deoxy form).
R (relaxed) state: High oxygen affinity (oxy form).
The T↔R transition explains cooperative oxygen binding.
Hb also functions in nitric oxide metabolism, pH regulation, redox balance, and metabolic reprogramming.
Differentiate between haemoglobin and myoglobin.
Myoglobin: Monomeric, stores oxygen in muscle.
Haemoglobin: Tetrameric, transports oxygen in blood. Both contain heme prosthetic groups but differ in structure, subunit interaction, and cooperative binding behaviour.
Outline the basic structure of immunoglobulins (Igs).
Immunoglobulins are Y-shaped glycoproteins composed of:
2 Heavy (H) chains and 2 Light (L) chains, linked by disulfide bonds.
Each chain has constant (C) and variable (V) domains.
Antigen-binding sites (Fab regions) lie at the variable domains’ N-termini, involving residues from both H and L chains.
Sequence diversity in variable regions determines antigen specificity.
What is the Immunoglobulin Superfamily (IgSF)?
The IgSF comprises a large group of proteins that share the Ig domain structure—a β-sandwich of two sheets linked by a disulfide bridge. Originally discovered in antibodies, these domains now appear in diverse proteins, including:
Antigen receptors and co-receptors
Cell adhesion molecules
Cytokine receptors
Antigen-presenting molecules
Intracellular muscle proteins . They mediate cell–cell recognition and immune communication across species.
Define the Fab and Fc regions of an antibody.
Fab (Fragment antigen-binding): N-terminal region containing variable domains responsible for antigen recognition.
Fc (Fragment crystallisable): C-terminal constant region responsible for effector functions (e.g., complement activation, Fc receptor binding). The Fc name derives from its crystallisable nature due to conserved sequence homology.
Describe the structural organisation of immunoglobulin domains.
Each Ig domain consists of two β-pleated sheets linked by a disulfide bridge between conserved cysteines. Each domain (~110–130 amino acids; 12–13 kDa) forms a compact β-sandwich.
Light chains: 1 variable (V_L) + 1 constant (C_L) domain.
Heavy chains: 1 variable (V_H) + 3–4 constant (C_H) domains. Constant domains hold H-chains together and mediate effector functions, while variable domains confer antigen specificity.
What are κ (kappa) and λ (lambda) light chains?
Antibody light chains exist in two types—κ and λ—defined by constant region sequence differences. Each antibody molecule carries only one type of light chain. The light chain’s N-terminal half is variable, while the C-terminal half is constant.
Explain how heavy-chain differences define antibody isotypes.
Five classes of heavy-chain constant regions exist:
γ → IgG
α → IgA
μ → IgM
δ → IgD
ε → IgE
Each antibody contains one type of heavy chain, defining its isotype and functional properties.
IgM (pentameric) contains 10 heavy (μ) and 10 light chains linked by a joining (J) chain; others are monomers.
What happens when immunoglobulins are enzymatically cleaved?
Papain cleavage: Produces two Fab fragments (antigen-binding) and one Fc fragment (effector).
Pepsin cleavage: Produces one F(ab′)₂ fragment retaining both antigen-binding sites but lacking Fc functions.
Clinical uses: Fab fragments — e.g., Digoxin immune Fab therapy. F(ab′)₂ fragments — antivenom treatment. Cleavage separates antigen recognition from immune effector activity.
Describe the domain structure of immunoglobulins.
H and L chains contain homologous domains (~110 residues each) connected by intrachain disulfide loops (~60 residues).
L chain: V_L + C_L.
H chain: V_H + 3–4 C_H domains (CH₁–CH₄). Domains maintain a consistent folding pattern forming the characteristic Ig fold.
Explain the significance of variable (V) region domains.
V_H and V_L domains determine antigen specificity. Within these, three hypervariable (HV) regions, also known as complementarity-determining regions (CDRs), form the antigen-binding site. The intervening framework regions (FRs) provide structural support. CDR3 is the most variable and central to antigen recognition.
Describe the features of constant (C) region domains in immunoglobulins.
Constant regions exhibit limited variability within an isotype.
H-chain C regions (CH₁–CH₄) mediate effector interactions, complement fixation, and Fc receptor binding.
CH₂ and CH₃ are critical for immune signalling and transplacental antibody transfer (in IgG).
CH₄ (when present) contributes to polymerisation and secretion.
