Magnetically Aligned Lipid Bilayers with High Cholesterol for Solid-State NMR of Membrane Proteins

Abstract

Phospholipid bicelles are valuable membrane model systems to study membrane proteins by NMR and other physicochemical techniques. The range of bicelle compositions that are compatible with uniaxial alignment of the lipid bilayers in a magnetic field is still limited with regard to the addition of large amounts (>20%) of cholesterol and/ or sphingolipids. Here, we demonstrate that n-dodecyl-β-D-melibioside (DDMB), which was recently introduced as a detergent to produce sphingolipid−cholesterol-rich isotropic bicelles for solution NMR studies, can also be used to produce magnetically alignable lipid bilayers with high cholesterol content that are well suited for solid-state NMR of membrane proteins. Remarkably, DDMB enables the preparation of high q bicelles that contain 50% mol cholesterol while retaining their ability to form a stable, well-aligned liquid crystalline bilayer phase in a magnetic field. We show that the intact 46-residue membrane-bound form of Pf1 bacteriophage coat protein and a truncated construct of the membrane protein Vpu from HIV-1 (residues 2−30) in DDMB bicelles are well aligned and undergo fast and uniaxial rotational diffusion about the bilayer normal, similarly to what is observed in other bicelle and macrodisc systems. We also demonstrate a spectroscopic method that measures the increase in the thickness of DMPC bilayers that results from the addition of cholesterol, using the PISA-wheel spectral patterns of trans-membrane helices as a molecular goniometer. For example, we find that the hydrophobic thickness of DMPC bilayers is increased by approximately 2.5 Å in the presence of 35% mol cholesterol.

Peptoid-based macrodiscs of variable lipid composition for structural studies of membrane proteins by oriented-sample solid-state NMR

“…The bilayer milieu is now believed to be composed of heterogeneous lipid domains, called lipid rafts ( Simons and Toomre, 2000 ), which give rise to different bulk bilayer properties such as viscosity and elasticity. Recently, DMPC-based bicelles rich in cholesterol ( De Angelis et al, 2022 ) and cholesterol and sphingomyelin ( Hutchison et al, 2020 ) have been introduced for structure–function studies of membrane proteins by solid-state and solution NMR. Despite their utility for protein structure determination, however, the presence of detergents may influence the structure and function of the embedded membrane proteins.…”