Synthesis, Characterization, and Nanodisc Formation of Non‐ionic Polymers**

Abstract

Although lipid nanodiscs are increasingly used in the structural studies of membrane proteins, drug delivery and other applications, the interaction between the nanodisc belt and the protein to be reconstituted is a major limitation. To overcome this limitation and to further broaden the scope of nanodiscs, a family of non-ionic amphiphilic polymers synthesized by hydrophobic functionalization of fructo-oligosaccharides/inulin is reported. We show the stability of lipid nanodiscs formed by these polymers against pH and divalent metal ions, and their magnetic-alignment properties. The reported results also demonstrate that the non-ionic polymers extract membrane proteins with unprecedented efficiency.

Saponins Form Nonionic Lipid Nanodiscs for Protein Structural Studies by Nuclear Magnetic Resonance Spectroscopy

“…This is consistent with observations of nanodiscs formed from other belt molecules. 12,15 We then performed temperature-dependent 31 P (Figure 1) and 14 N (Figure 2) solid-state NMR experiments under static conditions to assess the magnetic alignment properties of nanodiscs formed with different DMPC/saponin mass ratios. At 293 K, a single peak appeared near −1 ppm (referenced to H 3 PO 4 ) in the 31 P NMR.…”

“…To demonstrate the utility of saponin nanodiscs for protein structural studies, we used purified saponin nanodiscs as an alignment medium for the measurement of RDCs from 15 Nlabeled cytochrome c. We performed 2D 1 H− 15 N in-phase antiphase HSQC (IPAP-HSQC) at 32 °C to measure NH (scalar ± dipolar) coupling values (Figure 6) in an anisotropic saponin nanodisc-based alignment medium. These couplings were compared to previously measured cytochrome c isotropic J NH couplings to determine the RDCs.…”

“…8 It is likely that higher polydispersity in the saponin nanodiscs resulted in a lower order parameter than that of the polymer nanodiscs and that this lower order parameter caused the measured RDCs to be of a smaller magnitude. The lower correlations then stemmed from RDCs which were small relative to 15 N spectral line widths.…”

“…For example, electrostatic interactions due to highly charged belts have been reported to disrupt functional reconstitution of membrane proteins. 14,15 As such, there is considerable interest in the development of uncharged belt molecules to study complexes of oppositely charged proteins such as the REDOX complexes. But there is a general lack of belt molecules with net neutral charges, and most of those that exist are zwitterionic detergents, such as DHPC and DPS, which are known to denature some membrane and solution-phase proteins.…”

“…The first reported synthetic nanodisc was constructed from a membrane scaffold protein (MSP) belt and there are now a wide variety of peptides, synthetic polymers, and detergents reported to act as nanodisc belts. − It is important to carefully select an appropriate belt that maintains native structure, dynamics, and function for protein structural studies. For example, electrostatic interactions due to highly charged belts have been reported to disrupt functional reconstitution of membrane proteins. , As such, there is considerable interest in the development of uncharged belt molecules to study complexes of oppositely charged proteins such as the REDOX complexes. But there is a general lack of belt molecules with net neutral charges, and most of those that exist are zwitterionic detergents, such as DHPC and DPS, which are known to denature some membrane and solution-phase proteins. − Nonionic surfactants are also uncharged and have been shown to be less perturbative to membrane protein structure than charged and zwitterionic surfactants and as such are attractive targets to find nonionic, nondenaturing belt molecules …”