Sensitive Morphological Characterization of Oriented High‐Density Lipoprotein Nanoparticles Using ³¹P NMR Spectroscopy

Abstract

The biological function of high‐density lipoprotein (HDL) nanoparticles, the so‐called good cholesterol that is associated with a low risk of heart disease, depends on their composition, morphology, and size. The morphology of HDL particles composed of apolipoproteins, lipids and cholesterol is routinely visualised by transmission electron microscopy (TEM), but higher‐resolution tools are needed to observe more subtle structural differences between particles of different composition. Here, reconstituted HDL formulations are oriented on glass substrates and solid‐state 31P NMR spectroscopy is shown to be highly sensitive to the surface curvature of the lipid headgroups. The spectra report potentially functionally important differences in the morphology of different HDL preparations that are not detected by TEM. This method provides new morphological insights into HDL comprising a naturally occurring apolipoprotein A‐I mutant, which may be linked to its atheroprotective properties, and holds promise as a future research tool in the clinical analysis of plasma HDL.

Analysis of the orientation of cholesterol in high-density lipoprotein nanodiscs using solid-state NMR

“…For NMR analysis, the particles were precipitated from aqueous solution by polyethylene glycol (PEG) as described previously. 31 Visualisation by transmission electron microscopy (TEM) confirmed the expected rHDL discoidal morphology with a mean diameter of 12.6 nm (2.3 nm SD)…”

“…The rHDL particles were prepared as described previously 31 using the widely established detergent mediated sodium cholate dialysis method. 30 Briefly, a thin film of POPC : [2,3,4- 13 C 3 ]cholesterol at a 10 : 1 (M/M) molar ratio was prepared as above.…”

“…The rHDL particles were precipitated in a ≥ 48 hour incubation at 4°C with 24 % (w/v) PEG-6000 in rHDL buffer, pH 7.4. 31,41 The precipitated particles were pelleted at 4°C by ultracentrifugation at 41 krpm for…”

“…The rHDL particles were precipitated in a Z48 hour incubation at 4 1C with 24% (w/v) PEG-6000 in rHDL buffer, pH 7.4. 31,41 The precipitated particles were pelleted at 4 1C by ultracentrifugation at 41k rpm for 2 hours in an SW55 Ti rotor (Beckman Coulter). The PEG-precipitated rHDL was transferred to a 3.2 mm diameter MAS rotor and stored at 4 1C.…”

“…[9][10][11][12] Wide-line and oriented 2 H NMR of deuterated cholesterol has been used extensively to study the effects of cholesterol on membrane ordering, 13 membrane deformation 9 and dynamics. 14,15 Additionally, natural-abundance 13 C-1 H NMR and 31 P NMR have been used to study the segmental motions of cholesterol and phospholipids 16 and the effects of cholesterol on the phase behaviour of lipid bilayers. 17,18 Cholesterol adopts, on average, an upright orientation approximately parallel with the lipid bilayer normal with the 3-OH group situated in the vicinity of the phospholipid glyceryl backbone and pointing toward the interface between the bilayer surface and the aqueous phase.…”