An acid-compatible co-polymer for the solubilization of membranes and proteins into lipid bilayer-containing nanoparticles

Positively charged poly(styrene-co-maleimide) extracts functional membrane proteins into nanodiscs, overcoming some limitations of current nanodisc technology.

was heated under reflux at 125 ℃ for 2 hours until the solution clarified. Solubilized copolymer was precipitated after cooling by lowering the pH to 5.0 with the drop-wise addition of concentrated hydrochloric acid and then washed 3 times in ultra-pure water. Washed, precipitated SMA was re-dissolved in 0.6 M NaOH overnight and the precipitation and washing procedure repeated. Finally, SMA was dissolved in a minimal volume of 0.6 M NaOH, pH adjusted to 8.0 and lyophilized. SMA stocks were made directly from the dried powder. Nanodiscs were left at 25 ℃ for at least 16 hours to ensure equilibration prior to use.

Negative Stain Transmission Electron Microscopy (TEM)
400 mesh carbon coated Cu grids were glow discharged twice for 15 seconds with a 15 second pause between charges. SMILP solutions were diluted to 0.1 mg/mL DMPC, 0.03 % w/v SMI in 50 mM NaOAc, 200 mM NaCl, pH 5 and centrifuged at 16,000 × g for 10 minutes to remove particulate contaminants. 5 µL of diluted SMILP solutions were added to the glow discharged grids and allowed to adsorb for 1 minute. The grids were washed three timed with ultra-pure water and stained twice successively for 1 minute with 1 % w/v phosphotungstic acid. Excess liquid was removed from the grids at each stage by blotting with filter paper.
Samples were imaged on a Tecnai T20 twin-lens transmission electron microscope (FEI, Cambridgeshire, UK) operating at an accelerating voltage of 200 kV. Images were recorded at a magnification of × 62,000 at -1.5 µm under focus. Images were recorded on an Eagle 4k × 4k CCD camera (FEI, Cambridgeshire, UK). Subsequent image analyses were carried out in ImageJ (FIJI). A total of 1038 particles were analyzed from micrographs where staining was of high quality to allow for reliable particle picking. 31 P NMR Spectroscopy 31 P NMR was carried out essentially as previously described, 2 but with the following modifications. SMI and DMPC mixtures were prepared as described above in 50 mM sodium acetate, 200 mM NaCl, pD 5.0 using DMPC concentrations of 7.50, 5.00, 2.50 and 1.25 mM. The M n value for SMA2000I was used to calculate molar concentrations of SMI, as the distribution was assumed to remain unchanged throughout the solubilization procedure described above. 31 P NMR spectra were recorded at 298 K using an Avance III 400 MHz NMR spectrometer (Bruker, UK) operating with an excitation frequency of 161.98 MHz using 1 H-decoupling. 256 scans were recorded per measurement over a sweep width of 32051 Hz, with an acquisition time of 1.022 s, a pre scan delay of 6.5 s, a relaxation delay of 5 s and a pulse width of 7.25 µs. Spectra were referenced to an external standard of 85 % H 3 PO 4 in 10 % D 2 O to correct for any changes in field strength between measurements.

Thermodynamic Calculations
The thermodynamic analysis performed is based on the work of Vargas et al. 3 , and has been described by them and us 2 previously. Briefly, to obtain saturation and soluilization breakpoints (c S SAT and c S SOL , respectively) the 31 P-NMR peaks were integrated using TopSpin software (Bruker, UK) and the absolute integrals normalized to the largest and smallest value in each data set. Normalized integrals were then averaged from three independent measurements, plotted as a function of SMI molar concentration and fit to the scenario described previously [2][3][4][5][6] . This fitting procedure yields c s SAT and c S SOL breakpoints, ie, the concentration of SMI required for the onset and completion of solubilization at each DMPC concentration. Plotting these breakpoints as molar concentrations of SMI against DMPC yields the phase boundary lines. The gradient of each of these lines describes the molar ratio of SMI to DMPC required for the vesicle saturation, , , and solubilization, . These molar ratios allow the calculation of partitioning coefficients for , SMI and DMPC which in turn allow for the calculation of the free-energy of transition of both DMPC, , and SMI, , transitioning from a vesicle/aggregate to a nanodisc. A detailed description of this analysis has been published previously 3 .

Dynamic Light Scattering
Dynamic light scattering experiments were performed as previously described 2 . In brief, samples were loaded into 45 µL quartz cuvettes with a 3 × 3 mm light path (Hellma Analytics, Germany). Measurements were taken using a Zetasizer Nano S (Malvern Instruments, Worcestershire, UK) equipped with a He-Ne laser at 633 nm with a detector angle of 178° relative to the incident beam. All measurements were performed after equilibrating the sample at 25 °C for 60 seconds. Each sample measured 3 times with the attenuator position automatically optimized for size determination. Each measurement consists of 11 scans of 10 seconds. Freeze-thaw stability was performed by flash freezing the nanodisc solution in liquid N 2 for 5 min and then thawing at room temperature before taking the sample to load into the cuvette. Temperature stability was performed by increasing the temperature from 4 to 80 °C in 1°C increments. The samples were measured at each temperature after equilibrating at that temperature for 2 minutes. Data analysis was performed after taking into account the viscosity and refractive index of all buffer constituents by fitting a non-negatively constrained least squares function to the measured autocorrelation function. This gives an intensity weighted particle distribution assuming spherical particles, which is converted to a volume weighted particle size distribution using Mie scattering theory 7,8 . A volume weighted PSD takes into account the increased scattering of light by larger particles to give a more realistic representation of the particles present. Cumulant analysis was also performed to obtain the Z-average diameter and polydispersity index 9 . The polydispersity index (PDI) is defined as the square of the ratio of the peak value to the width of the Gaussian distribution obtained from cumulant analysis.

Small Angle X-ray Scattering (SAXS)
SMILP samples were prepared as described above, at a final solution concentration of 1.5wt% SMI, with 3, 5 or 7 mg/ml DMPC in a pH 5 acetate buffer containing 0.2 M NaCl. Samples were not gel filtered prior to measurement. SMILP solutions, a solution of the SMI polymer alone at 1.5 % (w/v) in the same buffer and the buffer were placed in a 96-well plate at 25 C, and loaded into the Arinax BioSAXS robot sample changer on the SAXS beamline B21 at Diamond Light Source.
Solutions were measured using the standard beamline configuration, at 12.4 keV, in a 1mm diameter quartz capillary, that was automatically washed, dried, and flushed with buffer before each measurement. Measurements were taken as 60 frames of 1 second using a Pilatus 2K detector.
The buffer solution was measured before and after each sample solution in the same capillary, and background scattering was subtracted from the data. Data was measured over a q range of 0.008 to 0.4 Å -1 , calibrated using silver behenate and reduced using the data reduction pipeline in DAWN 10 .

Fitting of SAXS Data
The During fitting as many parameters as possible were pre-calculated and held, to reduce the number of free parameters in the model. The scattering length density of the SMI polymer, the lipid tails, lipid headgroups and the solvent were all calculated and held during fitting (see Table   S1) while the lipid tail thickness and headgroup thickness were set to values previously determined for DMPC bilayers 14 . The background was set using the observed experimental background at high Q. The fitted parameters were therefore the scale factor for the ellipsoid model corresponding to the free polymer, and for the bicelle model, the scale factor, the core radius, the polydispersity in the core radius, the belt thickness and belt region scattering length density were fitted. Errors in For radioligand binding assays, cells were seeded at a density of 5 x 10 5 cells/100 mm dish and transfected after 48 h. Transfection was essentially as described previously 18 . Briefly, cells were transfected with either human A 2A R or human V 1a R cDNA in pcDNA3.1(+) using a mixture of 5 µg DNA, 60 µl polyethyleneimine (10 mM) and 1 ml 5 % glucose solution, which was incubated for 30 min at room temperature before addition to an appropriate final volume of full media.

SUPPORTING DATA
Figure S1: Volume weighted particle size distribution data measured using DLS at SMI concentrations below the polymer concentration required to initiate solubilization of 7.5 mM DMPC, c S SAT , the saturation boundary. Before the addition of SMI, DMPC is present as small unilamellar vesicles. Upon addition of low concentrations of SMI below c S SAT (0.1 mM SMI), SMI induces aggregation of DMPC. As c S SAT is surpassed, the distribution shifts towards smaller diameters. As c S SOL is approached, a sharp peak of a smaller hydrodynamic diameter (D h ) appears as the proportion of DMPC present as SMILPs increases.
[DMPC] / mg/mL  Table S1. Fitting parameters used to fit SAXS data of SMILPs to a model of a poly-core bicelle.
To account for free polymer in solution, this model was merged with that for an ellipsoid.
Parameters marked with * were fixed throughout the fitting procedure. Figure S3. DLS particle size distribution data for SMILP nanodiscs subjected to successive freeze-thaw cycles. After 10 freeze-thaw cycles, only a small shift of the distribution was observed.
No large aggregates were observed, suggesting that SMILPs remain intact through multiple freezethaw cycles.