Real-time imaging of lipid domains and distinct coexisting membrane protein clusters

https://doi.org/10.1016/j.chemphyslip.2011.12.012Get rights and content

Abstract

A detailed understanding of biomembrane architecture is still a challenging task. Many in vitro studies have shown lipid domains but much less information is known about the lateral organization of membrane proteins because their hydrophobic nature limits the use of many experimental methods. We examined lipid domain formation in biomimetic Escherichia coli membranes composed of phosphatidylethanolamine and phosphatidylglycerol in the absence and presence of 1% and 5% (mol/mol) membrane multidrug resistance protein, EmrE. Monolayer isotherms demonstrated protein insertion into the lipid monolayer. Subsequently, Brewster angle microscopy was applied to image domains in lipid matrices and lipid–protein mixtures. The images showed a concentration dependent impact of the protein on lipid domain size and shape and more interestingly distinct coexisting protein clusters. Whereas lipid domains varied in size (14–47 μm), protein clusters exhibited a narrow size distribution (2.6–4.8 μm) suggesting a non-random process of cluster formation. A 3-D display clearly indicates that these proteins clusters protrude from the membrane plane. These data demonstrate distinct co-existing lipid domains and membrane protein clusters as the monofilm is being compressed and illustrate the significant mutual impact of lipid–protein interactions on lateral membrane architecture.

Highlights

► Formation, size and shape of lipid domains depend on the ratio of lipid classes. ► Simultaneous formation of distinct protein clusters and lipid domains was observed. ► The narrow protein clusters already form at low pressures and their size distribution varied weakly with concentration. ► Mutual interactions of lipids and proteins impact cluster and domains size and shape.

Introduction

Lipids play an important role in transport across and structure of biological membranes. Various properties like fluidity and permeability are determined by lipid and protein compositions (van Dalen and de Kruijff, 2004, Zachowski, 1993). In recent years, the lateral lipid organization (Anderson and Jacobson, 2002, Harder, 2003, Simons and Vaz, 2004) for various biological functions in mammalian systems has been highlighted (Simons and Ikonen, 1997). In vitro studies have shown that well-defined lateral lipid domains have a strong propensity to form (Prenner et al., 2007, Wang et al., 2001, Wang and Silvius, 2000), but much less is known about their interactions with membrane proteins.

Approximately one third of all proteins are integral membrane proteins (Smith et al., 2001). Although there are over 58,000 independent protein structures in the Protein Data Bank, only about 200 membrane protein structures have been solved (Watanabe and Inoko, 2009). In our study we focus on imaging and characterizing the impact of the membrane protein EmrE from Escherichia coli in lipid monolayer model systems.

EmrE is 110 amino acids in length and has been shown to adopt a variety of multimeric states (Bay et al., 2008). It was first identified in E. coli (Purewal et al., 1990) and allows the organism to be resistant to many lipophilic cations including DNA intercalating dyes and quaternary ammonium compounds (Bay et al., 2008, Paulsen et al., 1996, Schuldiner, 2009). The structure of EmrE has been investigated by various approaches (Winstone et al., 2005) and although some controversy remains in the literature regarding its structure (Schuldiner, 2007), one important aspect of the protein is its propensity to be active as an oligomer.

The lipid compositions between the inner and outer membranes of E. coli differ, with the latter containing 40% phospholipids and 60% protein. In the inner membrane, the two major phospholipids species are phosphatidylethanolamine (PE), and phosphatidylglycerol (PG) (van Dalen and de Kruijff, 2004). PE is a zwitterionic lipid and makes up 75–80% of the E. coli inner membrane phospholipids (Cronan, 2003, Dowhan, 1997). Some PE lipids, depending on their acyl chain length and saturation, pH, or hydration, adopt non-bilayer structures such as the HII phase (cross-sectional area of the head group is smaller than the area of the acyl chains) resulting in membrane curvature (Gruner et al., 1985, van Dalen and de Kruijff, 2004, van Voorst and de Kruijff, 2000). These phase modulating properties may influence protein function and could be indicative of PE's importance in membrane protein assembly and enzyme function (Dowhan, 1997). PG is an anionic lipid and accounts for approximately 20% of the E. coli inner membrane (Dowhan, 1997, van Dalen and de Kruijff, 2004). E. coli membrane studies have demonstrated that PG is essential and may segregate into distinct domains with specific lipid and protein composition (Vanounou et al., 2003). Accordingly, DMPE and DMPG are the lipids selected for our lipid monolayer model system.

Ultimately, it is important to understand the mutual impact of lipids and proteins on membrane architecture. In order to visualize the lateral organization of lipids and the presence of membrane protein aggregates, we used Brewster angle microscopy, which has been established as an important marker-free imaging tool for lipid and/or protein monolayers at the air–water interface (Hénon and Meunier, 1991, Hönig and Möbius, 1991).

In this paper we present an E. coli membrane model system in the absence and presence of the integral membrane protein, EmrE, at 1% and 5% (mol/mol) concentrations. We show the integration of the protein into the monolayer, as well as its affects on lipid domain properties. Many studies have examined lipids with peptides or proteins using this technology. However, peptides are quite small and the tested proteins have mostly been limited to hydrophilic or soluble ones (Fidelio et al., 1981, Fidelio et al., 1982, García-Verdugo et al., 2007). In addition the more hydrophobic surfactant proteins have been studied in great detail at the air–water interface (Cruz et al., 2000, Cruz et al., 2004, Taneva and Keough, 2000). Although these studies were relevant and important, the study of integral membrane proteins requires additional considerations on the stability of membrane proteins in lipid monolayers. Albeit there are potential reservations of studying an integral membrane protein by this approach, we give a brief overview of previous work to provide a rationale for our experimental design. Salesse and coworkers have provided a comprehensive and in-depth analysis of this aspect (Boucher et al., 2007). Using PM-IRRAS, monolayer studies have shown that protein secondary structure can be retained depending on the nature of the protein and the detailed experimental conditions. These authors established that the membrane receptor rhodopsin will need low temperatures (4 °C) and spreading at 5–10 mN/m to retain the native structure that was assessed by infrared spectroscopy in the monofilm (Lavoie et al., 2002). Nevertheless, rhodopsin denaturation may be prevented when the protein is spread in excess detergent (Salesse et al., 1990). In contrast, bacteriorhodopsin that is found as a 2-dimensional crystal in purple membranes (Lavoie et al., 1999) was shown to be stable after spreading at 0 mN/m and prolonged incubation (Lavoie et al., 1999). Moreover, others groups reported that the crystalline character of intact purple membranes was maintained at the air–water interface (Verclas et al., 1999). EmrE is a very stable and hydrophobic membrane protein that was found to be more resistant to denaturation than bacteriorhodopsin. The latter showed a 30% change to helical content in the presence of low 0.2% SDS concentrations, whereas the former does not denature under these conditions (Miller et al., 2009). Interestingly, EmrE shows limited changes in helicity only under the combined action of 10 M urea and 5% SDS (Miller et al., 2009). An additional advantage to using EmrE for these studies is that as it is so hydrophobic, it is soluble in chloroform: methanol solvent mixtures (Winstone et al., 2002). Imaging with Brewster angle microscopy provides a unique tool to study co-existing lipid and membrane protein domains, which in the case of EmrE under our experimental conditions were found in the center of the distinct lipid domains.

Section snippets

Purification of EmrE

Purification of EmrE was followed similar to Winstone et al. (2002). EmrE protein was expressed from E. coli strain LE392Δunc containing the expression plasmid pMS119EH with the cloned emrE gene positioned behind a tac-promoter. Cells were grown in 1 L Terrific Broth at 37 °C, harvested by centrifugation, and washed twice before they were lysed by two passes through a French press (16,000 psi) and subsequent centrifugation to collect the membrane fraction (Winstone et al., 2002). The preparation

Monolayer isotherms

Monolayers have been widely used to study lipid (Brockman, 1994, Hönig and Möbius, 1991) and peptide monofilms (Momsen et al., 1997). For our initial studies, monolayers serve as a suitable model system as they allow high compositional control. Moreover, the impact of lipid–protein packing on their interactions cannot be easily assessed by other methods (Maggio et al., 2008). Fig. 1 showed several differences between binary lipid and lipid–protein isotherms and these samples were compared. The

Conclusions

All of these studies have expanded our understanding of lateral membrane architecture and emphasize the importance of lipid–protein interactions whereby the current contribution characterizes the coexistence of distinct membrane protein clusters within well-defined lipid domains and the impact of proteins on lipid domains. Moreover, the narrow size distribution of these observed clusters did not change significantly with protein concentration and indicates a non-random clustering process. The

Acknowledgements

This work was supported by discovery grants from the Natural Sciences and Engineering Research Council of Canada to RJT and EJP. We acknowledge the expert technical assistance by the instrument manufacturer, Accurion Inc. (Göttingen, Germany).

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