What is protein oligomerisation?

Protein oligomerisation is the process by which two or more protein molecules, called monomers, associate into a larger complex known as an oligomer. The subunits can be identical, giving a homo-oligomer, or different, giving a hetero-oligomer. Oligomerisation is fundamental to how many proteins are built and how they work, and the oligomeric state a protein adopts is often the state in which it is biologically active. It is also written oligomerization.

Why does the oligomeric state matter?

The active form of many proteins, including important drug targets such as the HIV-1 protease, EGFR, HER2 and TNF-alpha, is an oligomer rather than a single monomer. The oligomeric state has direct consequences for drug discovery, because it shapes the stoichiometry of binding, the avidity of an interaction and the stability of the protein in solution. It matters just as much in formulation and bioprocessing, where a therapeutic protein can exist as a mixture of monomer, dimer and higher-order species and where only one of those forms may bind the target receptor. Knowing which species dominates, and under which conditions, is part of understanding both the biology and the developability of a protein. Source: Fidabio application notes Reversible oligomerization and FIDA and Oligomerization in Plasma and Buffer.

How is oligomerisation different from aggregation?

Oligomerisation and aggregation both describe proteins associating into larger species, but they are not the same thing. Oligomerisation is usually specific, reversible and functional: a defined number of subunits assemble into a defined complex, and the equilibrium shifts with concentration and conditions. Aggregation is usually non-specific and often irreversible, tends to follow misfolding or unfolding, and generally reduces function. The practical difference is one of order and reversibility, and FIDA tells them apart with a simple dilution series, because a reversible oligomer dissociates on dilution while an aggregate generally does not. For the non-specific case, see What is protein aggregation?

How is protein oligomerisation measured?

Protein oligomerisation is measured by following molecular size in solution, because an oligomer has a larger hydrodynamic radius than the monomer, so a size above the monomer is the signal that association has occurred. Flow Induced Dispersion Analysis (FIDA), an efficient, in-solution method for protein characterisation, measures the hydrodynamic radius directly, which is what makes it able to detect and quantify oligomerisation. Because FIDA works from first principles, the expected size of the monomer can be predicted from the protein's molecular weight, or from a known structure using the PDB correlator, and a measured size larger than that prediction points to oligomerisation. Since non-covalent oligomerisation is concentration dependent, FIDA confirms it by dilution: if the measured size falls as the sample is diluted, the association is reversible, and a full concentration series run with the complex dissociation (CapDis) workflow determines the oligomerisation K_D.

As a worked example, bovine beta-lactoglobulin, a well known homo-dimer, measured by FIDA at high concentration gave a hydrodynamic radius of 2.9 nm, corresponding to 36.3 kDa on the size to molecular weight relationship, close to the reported dimer mass of 36.6 kDa and consistent with a dimer. Lowering the pH shifted the equilibrium, favouring the dimer near mildly acidic pH and the monomer below pH 3, and a CapDis concentration series returned the dimerisation K_D. Because FIDA immobilises and separates nothing, the same measurement runs label-free with UV detection or, with a fluorescent label, directly in crude matrices such as plasma and cell lysate, consuming only 40 nL of the loaded sample per data point. Source: Fidabio application notes Reversible oligomerization and FIDA (beta-lactoglobulin) and Oligomerization in Plasma and Buffer (measurement in 90% plasma). See the sizing and aggregation readout.

Common questions

How do you confirm a protein is oligomerising rather than aggregating?

Measure the hydrodynamic radius with FIDA, then dilute the sample and measure again. A reversible oligomer dissociates as the concentration drops, so its FIDA-measured size decreases, whereas an aggregate generally does not, which lets FIDA separate the two in one dilution series. Source: Reversible oligomerization and FIDA.

Can FIDA measure the oligomerisation K_D?

Yes. In FIDA a concentration series analysed with the complex dissociation (CapDis) workflow monitors the oligomerisation equilibrium directly and returns the K_D. Source: Reversible oligomerization and FIDA.

Can FIDA measure oligomerisation in plasma or serum?

Yes. FIDA is matrix independent, so it follows oligomerisation label-free with UV detection or, with a fluorescent label, directly in crude matrices such as plasma and cell lysate. Source: Oligomerization in Plasma and Buffer.

What size change does FIDA read as oligomerisation?

A hydrodynamic radius, measured by FIDA, larger than the value predicted for the monomer from its molecular weight or from its structure using the PDB correlator.

Related resources

The application note Reversible oligomerization and FIDA works through label-free detection and oligomerisation K_D determination on beta-lactoglobulin, and Protein Oligomerization: Easy and Accurate Determination in Plasma and Buffer follows monomer, dimer and tetramer states in buffer and in 90% plasma. The infographic Understand the conditions that drive oligomerization summarises the conditions that shift the equilibrium.