A major challenge for biophysical studies of membrane proteins is obtaining stable, homogenous samples. Traditional detergent solubilization and liposome-based methods of reconstitution may lead to protein inactivation, heterogeneous and polydisperse sized particles, and sample aggregation. [1] While membrane scaffold protein (MSP) stabilized nanodiscs have facilitated the formation of monodisperse protein samples, [2] a drawback is the detergent-based preparation method. Here we present a physicochemical characterization of polymer-stabilized lipid particles termed Lipodisq, a novel nanosized lipid-based platform capable of incorporating membrane proteins. [3] The polymers used in the Lipodisq technology can solubilize commonly used lipids such as dimyristoylphosphatidylcholine (DMPC) without the use of detergents. The small size of Lipodisq (diameter of around 9-10 nm at pH 7.4) renders them potentially suitable for many biophysical methodologies, including electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) spectroscopies, electron microscopy (EM), and circular dichroism (CD) spectroscopy.DMPC and a polymer formed from a molar styrene to maleic acid ratio of 3:1 (termed 3:1 SMA, see Figure S1 in the Supporting Information) were used as a model system to generate Lipodisq particles. Their formation using 3:1 SMA and DMPC at a weight ratio of 1.25:1.0 was followed by dynamic light scattering (DLS) at 550 nm and 25 8C (Figure 1), resulting in solution clarification within a minute. Higher SMA to DMPC ratios of 3:1 can also be used, though the excess polymer may increase the total solution viscosity, while decreased amounts results in sub-optimal DMPC solubilization. The dynamic light scattering data indicate a monodisperse distribution of Lipodisq particles with an average diameter of 9 nm as previously reported, [3b] which is confirmed by negative stain transmission electron microscopy (TEM, Figure 1 C). Figure 1. Light scattering data and size of Lipodisq particles. Lipodisq particle formation was observed by measuring the light scattering of the resulting polymer-lipid solution at 550 nm (A; Abs = absorption).DLS data (B) suggest that the Lipodisq formed have an average diameter of 9 nm, whereas TEM images (C) reveal that the Lipodisq diameter varies between 5-15 nm.
Lipodisq‐Partikel – Polymer‐Lipid‐Komplexe – wurden mit einer detergentienfreien Methode hergestellt. Dimyristoylphosphatidylcholin(DMPC)‐haltige Lipodisq‐Partikel zeichnen sich im Vergleich mit einer DMPC‐Dispersion durch stärker geordnete Lipide aus. Die Styrol‐ und Maleinsäuregruppen des Polymers wechselwirken mit den lipiden DMPC‐Ketten in der Doppelschicht und mit den lipiden Kopfgruppen in der Liposdisc‐Peripherie (siehe Bild).
Understanding the role of specific bilayer components in controlling the function of G-protein coupled receptors (GPCRs) will be a key factor in the development of novel pharmaceuticals. Cholesterol-dependence in particular has become an area of keen interest with respect to GPCR function; not least since the 2.6Å crystal structure of the β2 adrenergic receptor revealed a putative cholesterol binding motif conserved throughout class-A GPCRs. Furthermore, experimental evidence for cholesterol-dependent GPCR function has been demonstrated in a limited number of cases. This modulation of receptor function has been attributed to both direct interactions between cholesterol and receptor, and indirect effects caused by the influence of cholesterol on bilayer order and lateral pressure. Despite the widespread occurrence of cholesterol binding motifs, available experimental data on the functional involvement of cholesterol on GPCRs are currently limited to a small number of receptors. Here we investigate the role of cholesterol in the function of the neurotensin receptor 1 (NTS1) a class-A GPCR. Specifically we show how cholesterol, and the analogue cholesteryl hemisuccinate, influence activity, stability, and oligomerisation of both purified and reconstituted NTS1. The results caution against using such motifs as indicators of cholesterol-dependent GPCR activity.
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