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Monitoring of SMALP-nanodisc formation by microfluidic diffusional sizing

Authors

Sebastian Fiedler, Monika Piziorska, Haris Choudhery & Sean Devenish

Styrene–maleic acid (SMA) copolymers have become popular tools in membrane-protein research as they extract both lipids and membrane proteins from cellular membranes while preserving the bilayer structure of the membrane in so-called nanodiscs. Microfluidic diffusional sizing (MDS) demonstrates how the SMA-to-lipid ratio controls styrene–maleic acid lipid particle (SMALP) size, which can be a crucial parameter in the characterisation of embedded membrane proteins. Moreover, MDS offers the opportunity to measure nanodisc size as well as protein complex formation using the same sample.

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Introduction

Nanodiscs are disc-shaped patches of lipid bilayers that are widely used to stabilise membrane proteins for functional and structural studies. A common method to prepare nanodiscs is based on the addition of styrene–maleic acid copolymers (SMAs) to intact cell membranes which spontaneously generates nanodiscs known as SMA–lipid particles.1

In this application note, SMALP-nanodisc are prepared from fluorescently labelled unilamellar vesicles composed of phosphocholine lipids, which are common in both prokaryotic and eukaryotic cell membranes. Microfluidic diffusional sizing (MDS) on the Fluidity One-W was used to observe the onset of SMALP-nanodisc formation at low SMA-to-lipid ratios. As the SMA-to-lipid ratio is increased, vesicles disintegrate, coinciding with the presence of large and potentially heterogenous SMALPs. At even higher SMA-to-lipid ratios, the average hydrodynamic radius of SMALPs shrinks by a factor of ~4.5 which considerably reduces the amount of embedded lipid. Such variations in nanodisc size greatly alter the environment surrounding embedded membrane proteins and thus need to be considered when preparing biophysical and biochemical applications. MDS is therefore an ideal tool for researchers studying or using SMALPs to enable tuning of SMALP size based on experimental requirements.


Methods

Preparation of fluorescently labeled lipid vesicles

Chloroform stock solutions of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-(dipyrrometheneboron difluoride)undecanoyl-sn-glycero-3-phosphocholine (TopFluor® PC; both Avanti Polar Lipids) were mixed to yield a TopFluor PC content of 1 mol%. From the mixture, chloroform was evaporated, dried lipid films were desiccated overnight and re-hydrated using PBS buffer (pH 7.4) at a total lipid concentration of 10 mM. To prepare fluorescently labelled large unilamellar vesicles (LUVs), 10 mM lipid (POPC/TopFluor PC 1 mol%) in PBS buffer was passed 30 times through a polycarbonate membrane with a pore size of 100 nm using an Avanti Mini Extruder and a syringe volume of 1 mL.

The resulting fluorescently labelled LUVs were analysed by dynamic light scattering yielding a Z-average hydrodynamic diameter of 118 ± 7 nm and a polydispersity index of 0.2 ± 0.08.

Preparation of SMALP-nanodiscs

Styrene–maleic acid (SMA 3:1, Polyscience) copolymer stock solutions were diluted in PBS (pH 7.4). Molar concentrations of SMA were calculated based on an average number-based molar mass of 4 kg/mol.2 To analyse nanodisc formation by Fluidity One-W, LUVs were mixed with SMA 3:1 at various ratios and incubated for at least 2 hours at a temperature of 21 °C. Samples were then measured on the Fluidity One-W using the 2-20 nm size range setting.


Results

On the microfluidic chip, fluorescence intensities of the undiffused material (Fundiffused), and the diffused material (Fdiffused), are recorded as the sample plug passes through the chip, resulting in fluorescence traces as shown in Figure 1. The fluorescence traces of intact LUVs yield signal ratios (SR), SR = Fdiffused /Fundiffused, of ~0.15 indicating low levels of diffusion. In contrast, fluorescence traces of identical lipid mixtures in the presence of SMA yield SR values of ~0.75 indicating higher levels of diffusion caused by conversion of the large LUVs to much smaller SMALP-nanodiscs.

Inactive LUVs graph (fig. 1A)

Figure 1: Fluorescence traces of intact fluorescently labelled LUVs (Rh range 50–60 nm based on DLS) in comparison with fluorescently labeled SMALP-nanodiscs (Rh range 5–20 nm). A) Intact LUVs composed of POPC and TopFluor PC (1 mol%) were measured at a total lipid concentration of 50 µM.

SMALP nanodiscs graph (fig. 1B)

Figure 1B) For nanodiscs, 50 µM of lipid (initially in LUVs) was mixed with SMA at a final concentration of 35 µM.

To analyse how the ratio of SMA to lipid affects SMALP nanodisc size, fluorescently labelled LUVs at three different concentrations of total lipid (25 µM, 50 µM and 100 µM) were mixed with increasing amounts of SMA. These three different experiments fall on a single curve when SR is plotted as a function of the SMA-to-lipid ratio (Figure 2).


At SMA/Lipid ≥0.08, an increase in SR indicates that SMA extracts lipids from LUVs leading to formation of smaller particles that coexist with intact LUVs. The point of complete LUV solubilisation is at SMA/lipid = 0.15, as determined previously by 31P NMR.2 However, as shown by our Fluidity One-W experiments, at this point of complete LUV solubilisation, the average Rh, of the SMALP nanodiscs is 20 nm, and this decreases as more SMA is added to a final, stable Rh of 4.5 nm (Figure 2).


Based on the measured Rh the radius of the nanodiscs, Rdisc, was determined3 assuming a POPC-bilayer thickness of 4 nm,4 revealing that Rh values of 20 nm and 4.5 nm result in Rdisc values of 26 nm and 5 nm, respectively. Such variability in Rdisc directly translates into the number of SMALP-embedded lipids.

SMALP-Lipid graph (fig. 2)

Figure 2: Ratio of fluorescence intensities in undiffused and diffused channels SR and hydrodynamic radii (Rh) of SMALP nanodisc formation from fluorescently labeled LUVs. Both SR and Rh show three independent experiments in which SMA was mixed with LUVs having total lipid concentrations of 25 µM (diamonds), 50 µM (triangles) or 100 µM (squares). Dashed red and blue lines guide the eye. Dotted grey line indicates the point of complete vesicle solubilisation (only nanodiscs present) based on 31P NMR taken from (2).

For example, in a larger SMALP with an Rh of 20 nm, there are 60 POPC lipid molecules between the centre and the edge of the nanodisc, whereas only 11 POPC molecules make up the radius of a nanodisc with an Rh of 4.5 nm (0.63 nm2 per POPC).4 Notably, the presence of a membrane protein would further reduce the number of nanodisc-embedded lipids which should be kept in mind if the lipid environment is a relevant consideration for downstream analysis.


Conclusion

Microfluidic diffusional sizing is an ideal tool to monitor SMALP nanodisc formation using minute amounts of sample.5 According to our data, SMALP nanodiscs vary in their average hydrodynamic radii from 20 nm to 4.5 nm depending on the SMA-to-lipid ratio. This change in size will significantly alter the lipid environment around any embedded proteins and so should be considered by researchers working on such systems.


In this study, fluorescently labeled lipids were used to observe SMALP-nanodisc formation. Notably, similar experiments could be performed using a fluorescently labeled protein as a probe. Importantly, such an approach would enable researchers to confirm nanodisc sizes as well as to measure complex formation of the embedded membrane protein using the same nanodisc sample.


References

  1. Dörr, J.M.; Scheidelaar, S.; Koorengevel, M.C.; Dominguez, J.J.; Schäfer, M.; van Walree, C.A.; Killian, J.A. The Styrene-Maleic Acid Copolymer: A Versatile Tool in Membrane Research. Eur Biophys J Biophy. 2016, 45, 3-21. (publication)
  2. Vargas, C.; Cuevas Arenas, R.; Frotscher, E.; Keller, S. anoparticle self-assembly in mixtures of phospholipids with styrene/maleic acid copolymers or fluorinated surfactants. Nanoscale. 2015, 7, 20685-20696. (publication)
  3. Glover, K.J.; Whiles, J.A.; Wu, G.; Yu, N.; Deems, R.; Struppe, J.O.; Stark, R.E.; Komives, E.A.; Vold, R.R. Structural evaluation of phospholipid bicelles for solution-state studies of membrane-associated biomolecules. Biophys. J. 2001, 81, 2163-2171. (publication)
  4. Kučerka, N.; Nieh, M.P.; Katsaras, J. Fluid phase lipid areas and bilayer thicknesses of commonly used phosphatidylcholines as a function of temperature. BBA-Biomembr. 2011, 1808, 2761-2771. (publication)
  5. Azouz, M.; Gonin, M.; Fiedler, S.; Faherty, J.; Decossas, M.; Cullin, C.; Villette, S.; Lafleur, M.; Alves, I.D.; Lecomte, S.; Ciaccafava, A. Microfluidic diffusional sizing probes lipid nanodiscs formation. BBA-Biomembr. 2020, 1862, 183215 (publication).


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