Microfluidic Diffusional Sizing (MDS) —A novel method for characterizing protein interactionsMaren Butz1, Sean Devenish1, Luca Groß1, Sebastian Fiedler1, Thomas Barnes1, Magdalena A. Czekalska2, Georg Meisl2

1: Fluidic Analytics Ltd, Cambridge, UK2: University of Cambridge, UK

Microfluidic diffusional sizing (MDS) is an in-solution technique that measures the hydrodynamic radius of a protein complex, this technique can be used to determine the binding affinity of protein interactions. MDS requires minimal sample

preparation, no surfaces and can be performed in biological mixtures.

Here we show current applications of MDS from the literature and our own work.

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What is MDS, how does it work?

Microfluidic Diffusional Sizing, MDS, exploits the properties of liquids in laminar flow to determine the size of proteins and peptides in solution. The binding affinity of a protein interaction can then be determined by measuring the average size of the protein as the concentration of its binding partner is increased. This can be plotted on a logarithmic scale to give a binding curve (concentration of binding partner against average hydrodynamic radius) which can then be used to determine the KD of the interaction.

In practical terms; a simple titration must be performed to create a series of solutions with increasing concentrations of binding partner, 5 µL of each solution is then pipetted onto a microfluidic chip and this is inserted into the reading instrument. Each solution must be tested and then the instrument will create a binding curve and determine the KD as well as the hydrodynamic radius of complex and unbound protein.

MDS allows protein interactions to be studied using small volumes with high sensitivity, in their native state without the use of a matrix or surface (Yates et al, Nature Chemistry, September 2015, 7, 802-809).

MDS in the literatureProtein-Lipid InteractionsThe interactions of α-synuclein with lipid particles were probed using multiple orthogonal techniques including MDS, using a Fluidity One instrument (Fluidic Analytics Ltd, Cambridge, UK). MDS revealed that synuclein stabilized lipid particles can bind further free synuclein on the lipid surface. The combined insights from using multiple techniques resulted in a detailed picture of the interactions.Falke et al., Chemistry and Physics of Lipids, March 2019, 220, 57-65

Understanding the role of phosphatidylinositol-4,5-bisphosphate (PIP2) has in the regulation of transient receptor potential ankyrin 1 (TRPA1) channel using MDS. The Fluidity One instrument (Fluidic Analytics Ltd, Cambridge, UK) was used to determine the kinetics of the interactions between two peptides (L992-N1008 and T1003-P1034) and model lipid membranes in the presence of PIP2. The paper demonstrates that the two peptides (L992-N1008 and T1003-P1034) interact with lipid membranes only if PIP2 is present and their affinities depend on the presence of calcium. The paper also shows that the putative phosphoinositide-interacting domain contributes to the stabilization of the TRPA1 channel gate.Macikova et al., The FEBS Journal, May 2019, 14931

OligomerizationThe oligomerization of SBD641, the substrate binding domain of Hsp70, is probed using MDS. The thermodynamic parameters governing the oligomerization show that structural constraints on the oligomer size are likely determined by specific molecular interaction modes at the interface. The authors note MDS provides a way to observe the behaviour in native-like conditions without the protein interacting with a surface or matrix which could influence self-assembly behaviour.Wright et al., Biochemistry, May 2018, 57 (26), 3641-3649

AggregationHere MDS is used to monitor nanobody binding to α-synuclein to monitor the formation of fibrils. Qualitative binding information is obtained from just microliters of sample.Zhang et al., ChemBioChem, October 2016, 17(20), 1920-1924

Case Study 2 : Binding affinity of serine protease thrombin using MDS

Figure 1-4: MDS uses steady state laminar flow to enable determination of KD1. To do this, a stream of fluorescently labelled protein (red

dots) is introduced alongside an auxiliary stream. 2. These streams flow in parallel and because there is no

convective mixing the only way protein can migrate into the auxiliary stream is by diffusion, the rate of which depends on the size of the protein. Small proteins will diffuse rapidly, and large proteins and aggregates more slowly.

3. At the end, the streams are re-split, and at this point the degree of diffusion is fixed. The quantity of protein in each stream is then determined by the fluorescence from the label. The ratio of the fluorescence between the two streams gives the protein's hydrodynamic radius (Rh). MDS can be used to measure proteins in buffer and in crude solutions like cell lysates or biological fluids, because only the labelled species is detected.

4. If the test is repeated using a mixture of labelled protein and unlabelled binding partner, it is possible to observe the degree of binding due to the change in average size. Only species including the labelled protein are detected and measured. Titrating the binding partner against the labelled protein gives a binding curve from which the KD value can be calculated. The hydrodynamic radius (Rh) for the unbound protein and protein complex can also be calculated.

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Case Study 1 : Protein-Lipid interactions assessed by MDS

The interaction of α-synuclein with lipid membranes is believed to be key to its function, yet these interactions can result in amyloid fibril formation – a hallmark of Parkinson’s disease.

Test MethodHere solutions were prepared with varying ratios of α-synuclein and small unilamellar vesicles (SUVs) in phosphate buffer. These were assessed by MDS (reporting size in nm, tested using the Fluidity One instrument) and circular dichroism, CD (reporting ellipticity, testing using the spectropolarimeter JASCO J-810 instrument).

ConclusionWhile similar results are obtained, the MDS data offers more detail – this showed that at higher lipid concentrations the protein molecules distribute across the SUVs, resulting in a slight decrease in size but no change in ellipticity.In this way MDS could offer additional insights compared to CD – but importantly it also had fewer sample requirements.

Furthermore, as it relies on size only, MDS could be used to assess interactions where there is no protein structure change (i.e. no change in ellipticity).

Here it is shown how microfluidic diffusional sizing (MDS) can be used to determine the binding affinity of protein-protein interactions. In this experiment the Fluidity One-W instrument (Fluidic Analytics Ltd, Cambridge, UK) to assess the binding of serine protease thrombin to two aptamers (HD22 and TBA).

While methods exist to determine KD experimentally, reviews remark that these often require a degree of expertise to collect reliable data1 and can have technique specific limitations. MDS can characterize protein interactions in vitro with no surfaces and no calibration required.

Test MethodA set of samples were prepared to assess the KD of each aptamer binding thrombin. For these the concentration of aptamer was held constant at 1 nM in each sample, while the concentration of thrombin was varied from 0 nM to 1630 nM, to reflect values above and below the expected KD. 5 µL of each sample was run on the instrument and a graph was generated of concentration of thrombin (nM) vs hydrodynamic radius (nm) – results can be seen in fig.5. Table.1 compares the measured KD with literature reported values.

ConclusionTwo pre-labelled aptamers were mixed with an unlabelled protein and using MDS the KD of each interaction was successfully calculated. The calculated KD values are in good agreement with previously reported values, and the difference in binding affinity is clearly visible.

The technique presents a simple means to experimentally determine KD, and crucially does so without the need to alter the molecules beyond addition of a standard fluorescent label to one binding partner. Measuring size gives confirmation of on-target binding, by quick comparison of the expected and observed size. The method has no surface fixing, and no complex preparation steps –allowing binding to be observed rapidly and in near-native conditions.

Table 1 – comparison of KD measured to that found in the literature2,3

Fig 5 – Logarithmic binding curve of thrombin vs HD22 and TBA

1. Vivoli et al, Journal of Visualized experiments, 2014, Vol, 91.2. Tasset et al, Journal of Molecular Biology, 1997, Vol. 272, pp. 688-698.3. Bock et al, Nature, 1992, Vol. 355, pp. 564-566.

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