Apolipoprotein-induced conversion of phosphatidylcholine bilayer vesicles into nanodisks

Abstract

Apolipoprotein mediated formation of nanodisks was studied in detail using apolipophorin III (apoLp-III), thereby providing insight in apolipoprotein-lipid binding interactions. The spontaneous solubilization of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) vesicles occured only in a very narrow temperature range at the gel-liquid-crystalline phase transition temperature, exhibiting a net exothermic interaction based on isothermal titration calorimetry analysis. The resulting nanodisks were protected from proteolysis by trypsin, endoproteinase Glu-C, chymotrypsin and elastase. DMPC solubilization and the simultaneous formation of nanodisks was promoted by increasing the vesicle diameter, protein to lipid ratio and concentration. Inclusion of cholesterol in DMPC dramatically enhanced the rate of nanodisk formation, presumably by stabilization of lattice defects which form the main insertion sites for apolipoprotein α-helices. The presence of fully saturated acyl chains with a length of 13 or 14 carbons in phosphatidylcholine allowed the spontaneous vesicle solubilization upon apolipoprotein addition. Nanodisks with C13:0-phosphatidylcholine were significantly smaller with a diameter of 11.7 ± 3.1 nm compared to 18.5 ± 5.6 nm for DMPC nanodisks determined by transmission electron microscopy. Nanodisk formation was not observed when the phosphatidylcholine vesicles contained acyl chains of 15 or 16 carbons. However, using very high concentrations of lipid and protein (> 10 mg/ml), 1,2,-dipalmitoyl-sn-glycero-3-phosphocholine nanodisks could be produced spontaneously although the efficiency remained low.

Comparative EPR studies on lipid bilayer properties in nanodiscs and liposomes

“…This feature is also relevant, because preparation of nanodiscs requires homogenous suspension of lipids and detergent in buffer, and cholesterol is known to form detergent resistant fractions with saturated lipids [40,41] thus preparation of nanodisc with high concentration of cholesterol can prove to be unattainable in standard systems. The solution to this problem can be the detergent free systems where up to 10 mol% of cholesterol was successfully incorporated into the DMPC based nanodiscs [4]. The tighter packing introduced into system by MSP protein, is also similar to effect of forcing hexagonal H II phase preferring lipids to form a bilayer.…”

“…MSP proteins are almost exclusively helical and amphiphilic, and stabilise lipid bilayer by forming belt-like antiparallel dimer of proteins [2,3]. Many membrane proteins were successfully reconstituted into nanodiscs and studied both structurally and functionally [1,[4][5][6][7][8], taking advantage of the precise control of the lipid environment, protein oligomeric state and access to both intra-and extracellular part of studied protein. As always in case of introduction of a new model experimental system a good understanding of its physical properties is required to evaluate its biological relevance.…”

Solubilization of lipids and lipid phases by the styrene–maleic acid copolymer

“…The average size of the SMALPs was estimated manually from 16 well-defined individual particles randomly located through the image based on their maximum diameter using Adobe Illustrator software (San Jose, CA). This procedure was used to avoid potential artifacts such as stain-induced particle aggregation or inhomogeneous particle staining (Zhang et al 2011; Wan et al 2011; Scheidelaar et al 2015). …”

A Step-by-step Method for the Reconstitution of an ABC Transporter into Nanodisc Lipid Particles

“…The nanodisc has been successfully combined with biophysical methods such SPR, ITC, and fluorescence spectroscopy [12][13][14][15][16] to help membrane research that is struggling with quantitative methodology. Recently, the nanodisc has been combined with quantitative mass spectrometry to help identifying membrane protein interactomes with better coverage and accuracy 17 .…”