SMA-Lipodisq nanoparticles, with one bacteriorhodopsin (bR) per 12 nm particle on average (protein/lipid molar ratio, 1:172), were prepared without the use of detergents. Using pulsed and continuous wave nitroxide spin label electron paramagnetic resonance, the structural and dynamic integrity of bR was retained when compared with data for bR obtained in the native membrane and in detergents and then with crystal data. This indicates the potential of Lipodisq nanoparticles as a useful membrane mimetic.

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).

The significance of specific lipids for proton pumping by the bacterial rhodopsin proteorhodopsin (pR) was studied. To this end, it was examined whether pR preferentially binds certain lipids and whether molecular properties of the lipid environment affect the photocycle. pR's photocycle was followed by microsecond flash-photolysis in the visible spectral range. It was fastest in phosphatidylcholine liposomes (soy bean lipid), intermediate in 3-[(3-cholamidopropyl) dimethylammonio] propanesulfonate (CHAPS): 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bicelles and in Triton X-100, and slowest when pR was solubilized in CHAPS. In bicelles with different lipid compositions, the nature of the head groups, the unsaturation level and the fatty acid chain length had small effects on the photocycle. The specific affinity of pR for lipids of the expression host Escherichia coli was investigated by an optimized method of lipid isolation from purified membrane protein using two different concentrations of the detergent N-dodecyl-β-d-maltoside (DDM). We found that 11 lipids were copurified per pR molecule at 0.1% DDM, whereas essentially all lipids were stripped off from pR by 1% DDM. The relative amounts of copurified phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin did not correlate with the molar percentages normally present in E. coli cells. The results indicate a predominance of phosphatidylethanolamine species in the lipid annulus around recombinant pR that are less polar than the dominant species in the cell membrane of the expression host E. coli.

The membrane protein monoglucosyldiacylglycerol synthase (MGS) from Acholeplasma laidlawii is responsible for the creation of intracellular membranes when overexpressed in Escherichia coli (E. coli). The present study investigates time dependent changes in composition and properties of E. coli membranes during 22h of MGS induction. The lipid/protein ratio increased by 38% in MGS-expressing cells compared to control cells. Time-dependent screening of lipids during this period indicated differences in fatty acid modeling. (1) Unsaturation levels remained constant for MGS cells (~62%) but significantly decreased in control cells (from 61% to 36%). (2) Cyclopropanated fatty acid content was lower in MGS producing cells while control cells had an increased cyclopropanation activity. Among all lipids, phosphatidylethanolamine (PE) was detected to be the most affected species in terms of cyclopropanation. Higher levels of unsaturation, lowered cyclopropanation levels and decreased transcription of the gene for cyclopropane fatty acid synthase (CFA) all indicate the tendency of the MGS protein to force E. coli membranes to alter its usual fatty acid composition.