Application Notes

9 A P P L I C A T I O N N O T E PART 1: SIZING MEASUREMENTS WITH DLS Isothermal DLS analysis using the Prometheus Panta was applied to all samples to study the size of the different copolymer nanodiscs stabilized membrane proteins. The hydrodynamic radius (r H ) of the major peak in the size distribution analysis reveals the size of the most populated particles in the sample. The typical desired radius of these nanodiscs is below 20 nm. Consequently, copolymers with an r H value exceeding 20 nm (indicated in grey in Table 1) are initially excluded from further consideration. As an example, here we demonstrate the overlayed size distribution plots of WbaP nanodiscs in Fig- ure 1. Most of the samples exhibited a primary population of particle sizes with a peak around 10 nm (Figure 1A and Table 1). However, a few copolymer candidates demonstrated larger particle sizes of approximately 20-30 nm (Figure 1B and Table 1), which are unexpected for nanodiscs stabilizing a complex of the estimated size and therefore deselected from further study. In particular, sample 3-6, which consisted of particles with an r H of approximately 30 nm, did not exhibit any bands of protein in the SDS-PAGE result (Figure 2). Additionally, samples 7, 8 and 14 display a band at the molecular weight of the protein as observed in the SDS-PAGE (Figure 2). However, the DLS results of these three samples reveal the presence of a considerable number of larger particles, making them unsuitable for further structural assays (Figure 1B, SMALP BZ25, SMALP BZ30 and AASTY 11-50 (1) respectively). These observations demonstrate that DLS agrees with the results of SDS-PAGE in a copolymer screening workflow. Furthermore, it also exhibits the greater capabilities in the triage of additional candidates that may be overlooked by SDS-PAGE. In conclusion, isothermal DLS can be used as a key parameter to select optimal candidate copolymers for downstream applications. Despite each copolymer candidate displaying distinctive characteristics in terms of solubilization efficiency and the r H of the resulting nanoparticles, the Prometheus Panta system's low sample volume (10 µL) and short measurement time (50 seconds per sample), allows for rapid screening of dozens of copolymers at once. Optimal copolymers will produce nanoparticles with a uniform size distribution and low polydispersity. Protein-embedded SMALPS generally have a radius ~10nm, though there may be exceptions to this based on their cargo. Results

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