Spontaneous Lipid Nanodisc Fomation by Amphiphilic Polymethacrylate Copolymers

Abstract

There is a growing interest in the use of lipid bilayer nanodiscs for various biochemical and biomedical applications. Among the different types of nanodiscs, the unique features of synthetic polymer-based nanodiscs have attracted additional interest. A styrene–maleic acid (SMA) copolymer demonstrated to form lipid nanodiscs has been used for structural biology related studies on membrane proteins. However, the application of SMA polymer based lipid nanodiscs is limited because of the strong absorption of the aromatic group interfering with various experimental measurements. Thus, there is considerable interest in the development of other molecular frameworks for the formation of polymer-based lipid nanodiscs. In this study, we report the first synthesis and characterization of a library of polymethacrylate random copolymers as alternatives to SMA polymer. In addition, we experimentally demonstrate the ability of these polymers to form lipid bilayer nanodiscs through the fragmentation of lipid vesicles by means of light scattering, electron microscopy, differential scanning calorimetry, and solution and solid-state NMR experiments. We further demonstrate a unique application of the newly developed polymer for kinetics and structural characterization of the aggregation of human islet amyloid polypeptide (also known as amylin) within the lipid bilayer of the polymer nanodiscs using thioflavin-T-based fluorescence and circular dichroism experiments. Our results demonstrate that the reported new styrene-free polymers can be used in high-throughput biophysical experiments. Therefore, we expect that the new polymer nanodiscs will be valuable in the structural studies of amyloid proteins and membrane proteins by various biophysical techniques.

Hydrophobic Functionalization of Polyacrylic Acid as a Versatile Platform for the Development of Polymer Lipid Nanodisks

“…This was further confirmed by the carbonyl carbon resonance (≈185–180 ppm) and the appearance of new peaks in the aliphatic region (CH 2 ≈32 ppm, CH 3 ≈17 ppm, and quaternary C≈54 ppm) in 13 C‐CPMAS NMR spectra of the synthesized polymers as compared to PAA starting material (Figure c). 1 H‐NMR spectra were used to estimate the extent of functionalization by integration (Figure S1, Supporting Information) to be ≈40–50%, within the range of optimal hydrophobic to hydrophilic ratio as seen previously for nanodisks …”

“…The most common hydrophobic group used in SMA polymer nanodisks is the styrene moiety. It is known that styrene has a strong absorption in the UV region that can interfere with various biophysical techniques, and can also have nonspecific interactions with other aromatic groups from the protein . Recently, two styrene‐free polymers (diisobutylene maleic acid co‐polymer, DIBMA, and polymethacrylate) have been shown to form nanodisks; however there has been no systematic investigation comparing the effects of varying the hydrophobic functional groups used in the formation of nanodisks.…”

“…It is known that styrene has a strong absorption in the UV region that can interfere with various biophysical techniques, and can also have nonspecific interactions with other aromatic groups from the protein . Recently, two styrene‐free polymers (diisobutylene maleic acid co‐polymer, DIBMA, and polymethacrylate) have been shown to form nanodisks; however there has been no systematic investigation comparing the effects of varying the hydrophobic functional groups used in the formation of nanodisks. In this study, we employed a simple and robust modification method to vary the hydrophobic groups on a commercially available low molecular weight polyacrylic acid (PAA) which allowed us to observe how alkyl‐PAA affected the formation, stability, and other properties of nanodisks.…”

Semen‐derived amyloidogenic peptides—Key players of HIV infection

“…In particular, the recently developed polymers that vary in charge and can form lipid nanodiscs would be valuable to trap the intermediate structures of SEVI in different membrane compositions . In addition, the use of “styrene‐free” methacrylate polymer nanodiscs would enable high‐throughput screening of conditions by ThT fluorescence and CD experiments as demonstrated recently …”

Picturing the Membrane‐assisted Choreography of Cytochrome P450 with Lipid Nanodiscs

“…Among such peptides, we have successfully demonstrated that 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 22 A and 4F can be used for P450's metabolon reconstitution. [57] 3. 4F was developed by Kariyazono et al [51] and has been shown to overcome some of the technical limitations of 22 A, including high versatility in lipid composition and stability during the size-exclusion chromatography steps necessary for nanodiscs' purification.…”