2. Quaternary structure
• Refers to the organization of subunits in a protein with multiple subunits
• Subunits may be identical or different
• Associate to form dimers (TIM, HIV protease, DNA binding proteins), trimers (MS2 viral
capsid protein), tetramers (Haemoglobin, Proteasome, Bacterial photosynthetic reaction
center), etc.
• Subunits have a defined stoichiometry and arrangement
• Subunits are held together by weak, noncovalent interactions (hydrophobic, electrostatic)
• Typical Kd for two subunits: 10-8 to 10-16 M (tight association)
– Entropy loss due to association - unfavorable
– Entropy gain due to burying of hydrophobic groups - very favourable
3. Why do proteins attain quaternary structures?
• Morphological function:
Many proteins have functions that require creation of large, stable structures. These
include long, thin structural elements and large, hollow capsids and rings.
• Bringing catalytic sites together
More complex scaffolds may better support function e.g. by the introduction of a new
active site at the interface between subunits. It has been estimated that roughly one
sixth of oligomeric enzymes has an active site located at the inter-subunit interface.
• Stability
Larger proteins are more resistant to degradation and denaturation. Indeed, an increase
in oligomerization state is one of the protein stabilization strategies observed in
thermophilic organisms. Protein stability involves a fine balance between the
enthalpic stabilization by many weak nonbonded interactions and the competing effect
of various entropic factors of conformational mobility and solvation.
4. • Cooperativity (allostery):
Allostery and multivalent associations are other functions that create an evolutionary
force selecting large proteins with several identical active sites rather than monomeric
proteins with a single active site.
• Reduction of surface area:
In general, it is preferable to reduce the protein surface area that is exposed to solvent,
by creating a large protein with several identical active sites. Reduction of surface
area reduces the amount of solvent needed to hydrate proteins. The reduced surface
area provided by an oligomeric protein provides protection from degradation.
Reduced surface area also improves the diffusion of substrates to enzyme active sites.
5. Assume that the aqueous cytoplasm is composed entirely of 20 kDa subunits and that all
oligomers are spherical in shape. Thus, a monomer would be a sphere of radius 1.8 nm, a
dimer would have a radius of 2.2 nm, and so on.
Assume that the cytoplasm is 20% protein and that the bound water of hydration is ~1.4 g/g of
protein or a hydration shell ~0.6 nm thick.
If the aqueous cytoplasm is composed entirely of monomers, the hydrated proteins occupy
47% of the total volume, over twice the 20% volume occupied by the protein alone.
Assuming this same 0.6 nm layer of hydration, the volume of the hydrated protein drops to
40% for dimers, 35% for tetramers and 30% for dodecamers. Thus, oligomerization can
significantly reduce the amount of water bound to protein surfaces.
An analysis of surface area to volume ratio of oligomeric proteins
6. How is large proteins built?
The large proteins may be constructed in one of following ways:
1. As long single chains
2. As heterooligomers of several smaller chains
3. As homooligomers of identical chains
7. 1. Error control: By building a large complex from many small subunits, translation errors
may be reduced by discarding subunits with defects, providing an extra step for
proofreading.
2. Coding efficiency: Homooligomers provide a genetically compact way to encode the
information to build a large protein. It reduces the genetic space such as in viruses.
3. Genetic efficiency: Oligomeric proteins may be subjected to amplified evolutionary
pressures, as deleterious mutations may be more pronounced and thus removed sooner
from the gene pool. Conversely, the advantages of beneficial mutations may also be made
evident sooner.
4. Regulation of assembly: Large assemblies built of many identical subunits have
attractive regulatory properties, because they are subject to sensitive phase transitions. For
instance, actin is involved in many dynamic processes at the cell surface. A collection of
actin-binding proteins control the nucleation, growth, termination and disassembly of actin
filaments allowing fine spatial and temporal control.
What are the advantages of having multimers than large monomers?
8. Protein surface is irregular. What does enable proteins to bind specific molecules?
How does oligomerization of proteins occur in the cell?
Shape complementarity is necessary for large number of weak interactions and to maximize
the strength of interactions ((H-bonds and van der Waals).
9.
10. Finally, oligomerization can arise via fusion of a gene encoding a dimerization or
oligomerization domain, such as a coiled-coil domain, onto a previously monomeric protein.
13. Protein assemblies built of identical subunits are usually symmetric
The homooligomeric proteins found in modern cells are also highly symmetrical with soluble
oligomers forming closed complexes related by simple point groups and extended polymers
showing helical symmetry.
14. The human growth hormone-receptor complex
Asymmetric complexSymmetric complex
15. Why build symmetrical oligomeric proteins?
1. Stability of association: The stability of closed, symmetrical oligomers is a consequence
of two factors: (a) the specificity of protein-protein interfaces favors symmetrical
complexes, and (b) the maximum numbers of inter subunit interactions are formed in
closed complexes.
2. Finite assembly: Proteins must avoid unwanted aggregation. Point group symmetry
provides a method to create oligomers of defined copy number. Several disease states
seem to be the result of pathological aggregation of mutant proteins such as sickle-cell
anemia, Alzheimer’s disease and prion-related diseases.
3. Folding efficiency: Symmetric protein structures provide fewer kinetic barriers to folding
than do asymmetric structures.
16. Where does oligomerization of proteins occur inside the cell?
The primary sites for protein synthesis and folding are the cytosol and the endoplasmic
reticulum. Cytosolic proteins are synthesized, folded and oligomerized in the cytosol.
Membrane and secretory proteins are synthesized in the endoplasmic reticulum (ER) and
oligomerization typically occurs within the ER, although, in some cases, oligomerization
takes place in the intermediate compartment and Golgi apparatus.
17. What are the factors which can affect the oligomer formation?
1. Ligand binding: Many receptors undergo dimerization upon ligand binding.
2. Polymerization: Proteins such as actin can polymerize. Other proteins can polymerize
after undergoing a conformational change giving rise to amyloid fibrils.
3. Concentration: The oligomerization state of a protein depends on the concentration of
protein. At nM concentration the tendency to be in the monomeric state will be much
higher than in the μM or mM range.
4. Environmental condition: Weak associations may happen due to conditions such as
concentration, temperature, pH, solvent conditions (the ionic strength, metal cofactors and
effectors concentrations) and have higher Kd values in the μM or mM range.
5. Domain linker: it is known that variation of inter-domain linker lengths can result in
variations in oligomeric state. Some examples are the legume lectins, which can dimerize
by various modes as well as tetramerize, the cystine-knot growth factors and lumazine
synthase.
18. How to determine the oligomeric states of proteins experimentally?
In general, the following in vitro biophysical techniques can be used:
1. Size exclusion chromatography
2. Cross-linking
3. Analytical ultracentrifugation
4. Isothermal titration calorimetry
5. Mass spectrometry
6. Förster resonance energy transfer (FRET)
7. Scattering techniques
8. Yeast two hybrid assays
9. Fluorescence anisotropy
10. NMR spectroscopy
19. Characteristics of oligomeric interfaces
Interfacial residues tend to protrude from the surface of the protein and the interaction surface
tends to be circular in shape. Protein–protein interaction interfaces are relatively planar as are
many hetero-oligomer interfaces.
The buried surface area in obligate homodimeric proteins is usually greater than 1400 Å2.
In nonobligate complexes, the interface buried surface area is usually less than 2500 Å2,
whereas for weak and transient associations the buried surface area of the interface is less than
1000 Å2 .
It has been found that certain conserved residues or hot spots generally at the center of an
interface are responsible for most of the binding energy of an oligomeric interaction.
20. Inter-subunit interfaces are less non-polar, and have a greater proportion of hydrophilic and
polar residues, than a typical protein hydrophobic core. Approximately one-fifth of the
residues at oligomeric interfaces are polar, a greater proportion than is found in buried
hydrophobic cores.
Hydrogen bonds and salt bridges are important for the stabilization of oligomeric interfaces,
as suggested by the prevalence of polar hot spot residues. Early studies suggested that there is
about one hydrogen bond per 200 Å2 of subunit interface.
Oligomeric interfaces often have significant electrostatic and geometrical shape
complementarity that gives rise to the specificity of the interaction.
21. It has been calculated that the average oligomeric state of cellular proteins is tetrameric and a
survey suggests that 35% or more of the proteins in a cell are oligomeric.
Most oligomeric proteins are homo-oligomers.
Higher-order oligomers are less prevalent and a relatively small fraction of oligomeric
structures have odd numbered stoichiometries.
Most oligomeric proteins and essentially all homo-oligomeric proteins, are symmetrical. This
symmetry is most frequently cyclic, dihedral, or cubic.
Observations from the databases analysis
(A) Isologous dimer (B) heterologous tetrame (C)
Heterologous polymer.
22. Oligomeric state No. of
homo
oligomers
No. of
hetero
oligomers
Percent
Monomer 72 - 19.4
Dimer 115 27 38.2
Trimer 15 5 5.4
Tetramer 62 16 21.0
Pentamer 1 1 0.1
Hexamer 20 1 5.6
Heptamer 1 1 0.1
Goodsell and Olson, 2000, Annu. Rev. Biophys. Biomol. Struct. 29, 105-153.
Natural occurrence of oligomeric proteins in E. coli
Oligomeric state No. of
homo
oligomers
No. of
hetero
oligomers
Percent
Octamer 3 6 2.4
Nonamer 0 0 0.0
Decamer 1 0 0.0
Undecamer 0 1 0.0
Dodecamer 4 2 1.6
Higher oligomers 8 - 2.2
Polymers 10 - 2.7
23. So, do you think that all proteins should be multimer?
In most cases, evolution appears to drive proteins to larger size and thus to symmetric,
oligomeric complexes. In some specialized classes of proteins, however, functional
considerations have the opposite effect, favoring small, monomeric proteins:
1. Rapid diffusion: Cytochrome c, ferredoxin, plastocyanin, and other soluble electron
transport proteins must be small and streamlined to diffuse rapidly to their sites of action
in the crowded environment inside cells. Extracellular hydrolases, hormones, and many
toxins are small for the same reason.
2. Stability at low concentrations: Oligomeric proteins are unstable at very low
concentrations, so secreted proteins are commonly monomeric. Apparently, the disulfide
bridge serves primarily to hold the subunits together at the low concentrations found as the
toxin diffuses to its target.
24. Inappropriate quaternary interactions induce disease
Sickel-cell hemoglobin: Hydrophobic patch from the mutation in b2 subunit (Gln Val)
Thick fiber
25. 153 aa
Alpha – 144 and Beta – 146 aa
Hemoglobin Vs Myoglobin
Hemoglobin is a dimer or tetramer?
26. RMSD (α,β) of hemoglobin = 1.244 Å
(43% sequence identity)
RMSD (Hem_β, myoglobin) = 1.301
Å (no significant similarity)
RMSD (Hem_α, myoglobin) = 1.397 Å
(27% sequence identity)
27. Myoglobin (dimer) interface area = 399 Å2.
Why myoglobin itself is not multimeric? Why hemoglobin is not homotetramer?
Hemoglobin (alpha, alpha) interface area
= 276 Å2.
28. Hemoglobin (α,β) interface area = 814 Å2.Hemoglobin (β,β) interface area = 240 Å2.
Now, can you tell me whether hemoglobin is a dimer or tetramer?
