Understanding of a protein’s function is in many cases gained through an awareness of what it interacts with, with what affinity and at what time. Monitoring protein-protein interactions (PPIs) therefore can provide deep insights into everything from basic biology to the mechanisms of disease.
Limitations of existing systems for studying PPIs
There are a wide array of technologies used for studying PPIs - perhaps the two most widely used are Isothermal Calorimetry (ITC) and Surface Plasmon Resonance (SPR).
ITC is a true in-solution technique, in which the heat either released or absorbed in molecular binding during gradual titration is monitored. However, ITC generally requires large amounts of protein sample (typically 300-2000 µL at 10-50 µM concentration), and is dependent on the interaction of interest having a sufficiently large enthalpic change, which is not always the case.
SPR requires less material than ITC, but is not truly in solution, as it requires one of the protein binding partners to be tethered to a chip surface to monitor interactions through changes in plasmon resonance. Whilst highly sensitive, the requirement for surface functionalization means the analysis is not in solution. Consequently, surface attachment of proteins in the right orientation can be extremely challenging and in some cases lead to immobilisation artefacts and, where diffusion is slower than association, mass transport effects can occur.
The ideal therefore would be ITC quality in-solution data with SPR amounts of material. At Fluidic Analytics, this is what we’re working towards.
Using changes in hydrodynamic radius to study PPIs
The sizing of proteins using microfluidic diffusional sizing enables detection of PPIs in simple mixtures using latent labelling chemistry. This was initially demonstrated by Yates et al, using sizing to monitor a Parkinson’s-related immune complex formed by a nanobody and equimolar (5 μM) α-synuclein. This approach has since been developed into the technology that underpins the Fluidity One.
If fluorescently labelled samples are available, PPIs can be monitored in complex mixtures such as cell lysates (a; Arosio 2015, Zhang 2016).
In this case the change in size of the nanobody in the presence of α-synuclein allows the detection of the binding not only in homogeneous solution but also in cell lysate (b). Further to this, the nature of the interacting protein can be assessed, as is seen in the difference in size between nanobody bound to monomeric synuclein, and synuclein fibrils (d). The ability to measure the hydrodynamic radius of fluorescent proteins and biomolecules will be an enabling feature of the Fluidity One-W.
Critically all of these measurements are performed using proteins in solution in their native state, without the use of a matrix or surface, with high sensitivity and little volume. Ultimately this approach will enable kinetics and thermodynamics of binding reactions to be determined.
Accurate determination of stoichiometry for a protein complex is critical to our understanding how the complex will react to other proteins or small molecules. Here we look at how stoichiometry not only effects important viral protein-antibody interactions but also disease pathology.
Long form protein labelling protocol using Alexa Fluor 488 NHS Ester from Thermo Fisher Scientific. Includes troubleshooting, purification steps and an example calculation to help with concentration and volume calculations.
Lucie Machikova talking through her poster detailing the work carried out in her publication "Putative interaction site for membrane phospholipids controls activation of TRPA1 channel at physiological membrane potentials".
Macikova et al use Microfluidic Diffusional Sizing (MDS) along with other techniques to understand the role phosphatidylinositol-4,5-bisphosphate (PIP2) has in the regulation of transient receptor potential ankyrin 1 (TRPA1) channel. The Fluidity One was used to create binding curves of the interactions between two specific peptides (L992-N1008 and T1003-P1034) and model lipid membranes in the presence of PIP2.
Here are five of our favorite recent publications, highlighting both the role of protein-protein interactions in disease and how the characterization of these interactions may allow the development of new therapeutic strategies.
Here we assess the stoichiometry of α-synuclein binding to lipid vesicles by microfluidic diffusional sizing (MDS) on the Fluidity One and Circular Dichroism (CD). We find that the results obtained are consistent between the two techniques, but that MDS presents advantages in both speed and simplicity.
Here the Fluidity One-W is used to experimentally determine the KD of two protein aptamer interactions, with results in good agreement with literature values. Importantly the technique required no calibration, complex setup or specialist sample preparation. Furthermore, the interaction is assessed without immobilising or altering either molecule beyond the addition of a standard fluorescent label.
There are many proteins which interact with lipids in vivo, in complex relationships that have crucial impacts on organism health. These are often thought of as disease causing, but can also play roles in normal biochemical functions. Here are some examples of protein-lipid interactions from around the human body, which play a part in healthy activity in cells.
In this paper from Arosio et al microfluidic diffusional sizing (MDS) is used in combination with pre-labelled fluorescent biomolecules to size and quantify proteins in complex mixtures. Initially, MDS is compared to Dynamic Light Scatter (DLS) - and a good correlation is observed for monodispersed solutions.
Is it possible to determine binding kinetics with the Fluidity One?
It is not currently possible (or at least it is very challenging) to determine binding kinetics with the Fluidity One. However, we hope to address this need with our next instrument - the Fluidity One-W, which will enable detection of single (fluorescently labelled species) in a complex.
The Fluidity One
In-solution sizing and quantification of native protein in less than 10 minutes
Fluidity One applications
The speed, convenience and low sample consumption of the Fluidity One lends it to a number of routine lab applications