BPS2026 – Optimal control and characterization of peptoid-based lipodiscs for structure determination of membrane proteins

Magnetically aligned bilayers provide a planar, fully hydrated lipid mimetics for studying the conformations of membrane proteins in various lipid environments by solid-state NMR. Self-assembled, detergent-free macrodiscs composed of lipids and synthesized amphipathic peptoid belts yield magnetic alignment and NMR spectroscopic resolution comparable or superior to the widely used detergent-containing bicelles. Considerable improvement in both magnetic ordering and NMR resolution is achieved by mixing the zwitterionic lipids with the negatively charged lipids (DMPG or DMPA). Moreover, we demonstrate that highly aligned, detergent-free macrodiscs can be formed not only with DMPC but also with longer-chain lipids, such as DPPC, including unsaturated POPC lipids, and short-chain lipids such as DLPC. These macrodiscs stay highly aligned within a broad temperature range, up to 15 o C wide, as monitored by 31 P NMR at 11.7 T magnetic field. By controlling the lipid/peptoid molar ratios, lipodiscs of uniform diameters can be formed, varying from 20 to 40 nm in diameter as evidenced by their size distributions obtained by TEM microscopy. Interestingly, the macrodiscs can be formed with peptoids as short as 8-mers, which are barely sufficient to span the average hydrophobic lipid core. This leads to a hypothesis that peptoids may insert themselves perpendicularly to the membrane plane rather than laterally. As an illustration of their general applicability to membrane proteins, peptoid-based macrodiscs have been used to obtain high-resolution 2D solid-state NMR of 15 N labeled Pf1 coat protein (4.6 kDa) in DMPC and DPPC bilayers, and the much larger protein proteorhodopsin (PR, 29 kDa). NMR spectra of PR have exhibited better resolution as compared to bicelles, potentially due to the additional motional averaging arising from the uniaxial diffusion of the macrodisc as a whole around the membrane normal. Supported by NSF CHE 2404331.