Chemistry and Physics of Lipids 163 (2010) 117–126 Contents lists available at ScienceDirect Chemistry and Physics of Lipids journal homepage: www.elsevier.com/locate/chemphyslip Preparation of reconstituted acetylcholine receptor membranes suitable for AFM imaging of lipid–protein interactions Ngoc Vuong a,1 , John E. Baenziger a,∗ , Linda J. Johnston b,∗∗ a Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada b Steacie Institute for Molecular Sciences, National Research Council of Canada, 100 Sussex Drive, Ottawa, Ontario K1A 0R6, Canada article info Article history: Received 22 June 2009 Received in revised form 4 September 2009 Accepted 24 September 2009 Available online 2 October 2009 Keywords: Nicotinic acetylcholine receptor Atomic force microscopy Reconstitution Supported lipid bilayers Proteoliposomes Fluorescence abstract The nicotinic acetylcholine receptor (nAChR) has been reconstituted in POPC vesicles at high lipid–protein (L/P) ratios for the preparation of supported lipid bilayers with a low protein density for studies of protein–lipid interactions using atomic force microscopy (AFM). Initial reconstitutions using a standard dialysis method with bulk L/P ratios ranging from 20:1 to 100:1 (w/w) gave heterogeneous samples that contained both empty vesicles and proteoliposomes with a range of L/P ratios. This is problematic because empty vesicles adsorb and rupture to form bilayer patches more rapidly than do protein-rich vesicles, resulting in the loss of protein during sample washing. Although it was not possible to find reconstitution conditions that gave homogeneous populations of vesicles with high L/P ratios, an addi- tional freeze–thaw cycle immediately after dialysis did reproducibly yield a fraction of proteoliposomes with L/P ratios above 100:1. These proteoliposomes were separated by sucrose gradient centrifugation and used to prepare supported bilayers with well-separated individual receptors and minimal adsorbed proteoliposomes. AFM images of such samples showed many small features protruding from the bilayer surface. These features range in height from 1 to 5 nm, consistent with the smaller intracellular domain of the protein exposed, and have lateral dimensions consistent with an individual receptor. Some bilayers with reconstituted protein also had a small fraction of higher features that are assigned to nAChR with the larger extracellular domain exposed and showed evidence for aggregation to give dimers or small oligomers. This work demonstrates the importance of using highly purified reconstituted membranes with uniform lipid–protein ratios for AFM studies of integral membrane protein–lipid interactions. © 2009 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Atomic force microscopy (AFM) has been widely used to char- acterize the structures of peptides and proteins in supported phospholipid bilayers formed from both reconstituted and native membranes (Frederix et al., 2009). Ordered two-dimensional arrays of membrane proteins including rhodopsin, porins, bacterial hexag- onally packed intermediate layers, photosynthetic core-complexes and connexons are particularly amenable to high resolution AFM imaging (Arechaga and Fotiadis, 2007; Buzhynskyy et al., 2007; Fotiadis et al., 2003; Muller et al., 1997; Scheuring, 2006; Stamouli et al., 2003; Thimm et al., 2005). These studies have provided unprecedented structural data for supramolecular assemblies of membrane proteins in aqueous buffer under close to physiolog- ∗ Corresponding author. Tel.: +1 613 562 5800x8222; fax: +1 613 562 5251. ∗∗ Corresponding author. Tel.: +1 613 990 0973; fax: +1 613 952 0068. E-mail addresses: John.Baenziger@uottawa.ca (J.E. Baenziger), Linda.Johnston@nrc-cnrc.gc.ca (L.J. Johnston). 1 Tel.: +1 613 562 5800x8222; fax: +1 613 562 5251. ical conditions, where conformational changes and the effects of factors such as pH and ligand binding can be readily studied. The ability to obtain high lateral resolution, on the order of ∼1 nm, is critically dependent on obtaining samples with a high density of protein over a relatively large and flat membrane area. Reconsti- tution at such high protein density is still largely empirical and generally depends on removing detergent from a fully solubilized lipid/protein/detergent mixture. Interestingly, a recent report has shown that integral membrane proteins such as the bacterial light harvesting complex can be incorporated into existing lipid bilayers that are destabilized by detergent, providing a potentially useful approach for preparing arrays of protein for structural analysis from minimal amounts of protein (Milhiet et al., 2006). On the other hand, AFM studies aimed at probing protein–lipid interactions require the use of reconstituted samples with much lower levels of protein. Supported bilayers with high lipid–protein ratios are required for characterizing the distribution of individ- ual membrane proteins within the membrane plane and thus for probing the preference of proteins for various lipid environments. Supported films with high lipid–protein ratios are necessary for interrogating individual molecules, as for example in correlated flu- 0009-3084/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.chemphyslip.2009.09.003