Why protein stoichiometry matters 

Dr. Sean Devenish


June 1, 2024

The stoichiometry, or relative number, of proteins in a complex is one of those aspects of bioscience that is often hard to infer or quantify. However, it is increasingly seen as more and more important in drug discovery and disease pathology. The confirmed stoichiometry between two proteins has the potential to save precious time and resources for companies and academic institutes alike. Here’s why.

 Stoichiometry implies the overall function of a protein complex

Cellular processes are highly orchestrated and regulated events which are dependent on precise amounts of specific protein complexes, ligand, or enzymes. Determining the stoichiometry of a given complex, such as the number of antibodies binding to an antigen, helps us to understand the overall function of that complex. [1] This is crucial when the stoichiometry of a given complex can change depending on the concentrations of the constituent protein subunits in a reaction mixture or on states or modifications of proteins in the complex [2]. Thus, the stoichiometry of any given protein complex is a fundamental part of the description of that complex, and if we do not know the stoichiometry we cannot truly claim that we understand the interaction we are studying.

For example, the oligomeric state of a protein may determine whether it is pathological or not. Tumor necrosis factor alpha (TNF-α) is a prime example of this phenomenon, where the TNF-α monomer and trimer are inactive and active inflammatory cytokines, respectively.

Stoichiometry reveals how many antibodies would be required to neutralize a virus

Flaviviruses are positive-stranded RNA viruses that are endemic in many regions of the globe and cause significant morbidity and mortality in humans. Examples of this type of virus include: the mosquito-borne dengue virus, yellow fever virus, Japanese encephalitis virus, West Nile virus and Zika virus. It has been estimated that 390 million Dengue virus infections occur each year, with 3.6 billion people at risk of infection in more than 100 countries [3]. Flaviviruses cause a variety of disease manifestations including encephalitis and paralysis, massive hepatic injury, and haemorrhagic and plasma leakage syndromes. At present, there is no specific therapy to treat flavivirus infections; only vaccines have proven effective at reducing the impact of these viruses on public health.

Almost 100 years ago, the structure of antibodies was even known, interest in the mechanism and stoichiometry of “antibody-mediated neutralization” was already a topic of debate [4]. Early arguments focused on how many antibodies would be required to neutralize a virus. One popular concept was that viruses could be neutralized following the binding of a single molecule [5]. The alternative theory was the “multiple hit” model that assumed viral particles would be coated with antibodies and neutralized at a critical occupancy, estimated at 30 antibodies binding per virus particle [6].

The leading theory today is closer to the “multiple hit” theory; however, there are other factors to consider: resistant strains of flavivirus may express fewer epitopes or display them in an inaccessible manner. Other factors may limit epitope accessibility on the virion, such as steric constraints among densely arranged viral proteins [7], proximity to the viral membrane [8], or carbohydrates that shield antibody-binding motifs [9]. Importantly, it is now recognized that different antibodies possess different stoichiometric requirements for flavivirus neutralization, with some potent antibodies able to neutralize at low binding stoichiometries [10]. Further complicating matters, antibody binding at stoichiometries insufficient to neutralize the virus can lead to antibody-dependent enhancement (ADE) of infection, whereby antibody-mediated cellular uptake provides a secondary pathway for viral entry to, and infection of, cells [10].

Stoichiometry helps us understand causative agents of Alzheimer’s disease

Alzheimer’s disease is characterized by the presence of intracellular neurofibrillary tangles and extracellular plaques which consist primarily of clusters of Aβ peptide sequences [11]. Formation of these species starts with cleavage of amyloid precursor protein (APP) to form Aβ peptides, which form aggregates named oligomers. Importantly, these oligomers are able to catalyze the recruitment of further Aβ peptides and thereby grow to become Aβ fibrils that form plaques in the brain of Alzheimer’s disease sufferers.

Figure 1: Overview of how APP cleaved Aβ monomers aggregate to form Aβ fibrils. Adapted from Jureschi et al. 2019 [11].

Decades of research has now gone into trying to halt the formation of Aβ peptides and their oligomerization, however, only limited progress has been made despite this global effort.

The Aβ peptide seen in Alzheimer’s disease is not just produced in the central nervous system (CNS) but in most cells of peripheral tissues [12]. Human serum albumin (HSA) has been identified as a plasma protein which binds to Aβ protofibrils and clears it from the CNS and into peripheral tissues [13]. Therefore, it has been proposed that dysfunctions in plasma proteins can affect the clearance of Aβ protofibrils from the CNS and so lead to Aβ peptide plaque deposition within the brain [14].

A model of binding was proposed by Milojevic and Melacini, whereby HSA binds Aβ oligomers in a specific fashion. Each of the three domains of HSA is able to bind an Aβ oligomer with nM affinity. This binding interaction blocks interaction of the oligomers with further Aβ monomers and so inhibits further growth of Aβ fibrils and plaques [14].

Schematic model that summarizes the prevailing stoichiometries and affinities between HSA and Aβ protofibrils.

Figure 2: Schematic model that summarizes the prevailing stoichiometries and affinities between HSA and Aβ protofibrils. The curved dashed lines represent the possible steric hinderance between Aβ protofibrils binding to different domains. Adapted from Milojevic & Melacini [14]

Dynamic Light Scattering (DLS) was used to probe whether Aβ protofibrils are able to bind multiple HSA molecules. Given that HSA is able to bind three protofibrils, if the protofibrils themselves can bind multiple HSA molecules the expected outcome would be network formation. Extended incubation of mixtures followed by DLS analysis showed that discrete particles were formed but not extended networks, showing that each protofibril has only one binding site for HSA [14].


Whilst stoichiometry has been shown to play a pivotal role in many protein-protein interactions, much of the technology used to study stoichiometry is based on dated methods. Whilst many techniques exist to measure protein-protein interaction affinity, most do not also measure stoichiometry.

One recently developed method that measures protein-protein interaction affinity as well as stoichiometry is Microfluidic Diffusional Sizing (MDS). MDS has been used to measure stoichiometry of protein interactions in solution without surfaces, matrices or ionization, thus allowing scientists to observe the proteins in their native state.

For more information, watch the video below to learn how MDS is changing the way protein research is being carried out.


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