Designing a Useful Lipid Raft Model Membrane for Electrochemical and Surface Analytical Studies

Abstract

A model biomimetic system for the study of protein reconstitution or drug interactions should include lipid rafts in the mixed lipid monolayer, since they are usually the domains embedding membrane proteins and peptides. Four model lipid films composed of three components: 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), cholesterol (Chol) and sphingomyelin (SM) mixed in different molar ratios were proposed and investigated using surface pressure measurements and thermodynamic analysis of the monolayers at the air–water interface and imaged by Brewster angle microscopy. The ternary monolayers were transferred from the air–water onto the gold electrodes to form bilayer films and were studied for the first time by electrochemical methods: alternative current voltammetry and electrochemical impedance spectroscopy and imaged by atomic force microscopy. In excess of DOPC, the ternary systems remained too liquid for the raft region to be stable, while in the excess of cholesterol the layers were too solid. The layers with SM in excess lead to the formation of Chol:SM complexes but the amount of the fluid matrix was very low. The equimolar content of the three components lead to the formation of a stable and well-organized assembly with well-developed raft microdomains of larger thickness, surrounded by the more fluid part of the bilayer. The latter is proposed as a convenient raft model membrane for further physicochemical studies of interactions with drugs or pollutants or incorporation of membrane proteins.

Model Lipid Raft Membranes for Embedding Integral Membrane Proteins: Reconstitution of HMG-CoA Reductase and Its Inhibition by Statins

“…However, the overall expansion of the layer is a result of more fluid lipid film between more compact lipid domains with embedded protein in the lipid layer at the air–water interface. Moreover, the addition of the protein at a concentration of 0.02 mol % (vs lipids) causes a visible inflection in the isotherm at a surface pressure of 30 mN/m, which corresponds to the surface pressure value observed in real cell membranes. , This can also be visualized by the minimum in the C s –1 vs π plot (inset in Figure ), which is not observed in the isotherm recorded after spreading the empty liposomes and may result from the change in the organization of lipids in the presence of protein or from the changes in the HMG-CoA reductase conformation upon compression. , Thus, it can be concluded that some lipid–protein interactions occur at the physiological surface pressure of the membranes.…”

“…The layer formed during the spreading of liposomes at the air−solution interface was significantly less stable (Figure 3B) than the 1:1:1 DOPC/Chol/SM monolayers formed from the mixture of lipids in chloroform/methanol on the PBS buffer subphase 35,36 or on the Tris-HCl buffer subphase. However, the proteoliposome-derived layers were more stable in time (light blue), and the surface pressure remained constant for many hours, as in the case of the monolayer formed by spreading the sample of the ternary mixture of lipids in the organic solvent (black) (Figure 3B).…”

“…The characteristic parameters of these layers are listed in Table 1 . A reorganization of the structure of the 1:1:1 DOPC/Chol/SM (raft) monolayers 35 , 36 was observed at high surface pressures (π > 40 mN/m). The partial collapse typical of these types of layers is reflected by a decrease in the compressibility modulus ( C s –1 ) to zero at a surface pressure of 40 mN/m.…”

“…In this study, we demonstrate the successful incorporation of HMG-CoA reductase, the enzyme responsible for the synthesis of cholesterol, into liposomes. The lipid mixture used to prepare the liposomes and proteoliposomes containing HMG-CoA reductase was composed of DOPC/Chol/SM in a 1:1:1 molar ratio, which has been shown in our previous studies to be optimal for recreating a lipid system whose composition and surface properties mimic those of the lipid rafts of cell membranes. , The slightly larger than usually size of the liposomes (approximately 390 vs 200 nm) was attributed to possible repulsive forces between the different lipid components and the presence of double bonds in the DOPC molecules. The liposomes were characterized by a high degree of curvature, as shown by the fluorescence microscopy results.…”

Clusters of apoptotic signaling molecule-enriched rafts, CASMERs: membrane platforms for protein assembly in Fas/CD95 signaling and targets in cancer therapy

“…In this regard, lipid rafts are becoming promising therapeutic targets [1,[46][47][48][49][50][51]. Different approaches [6,[52][53][54][55] have contributed in the last two decades to unravel the biophysical, kinetic and mechanical properties of these membrane domains, including the use of: lipid raft model membranes (e.g. ternary lipid mixtures containing cholesterol, sphingomyelin and phosphatidylcholine derivatives in different molar ratios, to form liquid ordered phases, which may coexist with other rigid or fluid phases, depending on the temperature); giant unilamellar vesicles; giant plasma membrane vesicles and plasma membrane spheres; novel fluorescence lipid analogs; single-molecule imaging; and additional high temporal and spatial resolution techniques.…”