| Download | - View accepted manuscript: Preparation of Reconstituted Acetylcholine Receptor Membranes Suitable for AFM Imaging of Lipid-protein Interactions (PDF, 997 KiB)
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| DOI | Resolve DOI: https://doi.org/10.1016/j.chemphyslip.2009.09.003 |
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| Author | Search for: Vuong, Ngoc; Search for: Baenziger, John E.; Search for: Johnston, Linda J.1 |
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| Affiliation | - National Research Council of Canada. NRC Steacie Institute for Molecular Sciences
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| Format | Text, Article |
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| Subject | Nicotinic acetylcholine receptor; Atomic force microscopy; Reconstitution; Supported lipid bilayers; Proteoliposomes; Fluorescence |
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| 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 additional 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. |
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| Publication date | 2010-02-01 |
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| In | |
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| Language | English |
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| Peer reviewed | Yes |
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| NRC publication | This is a non-NRC publication"Non-NRC publications" are publications authored by NRC employees prior to their employment by NRC. |
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| NPARC number | 16086089 |
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| Export citation | Export as RIS |
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| Report a correction | Report a correction (opens in a new tab) |
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| Record identifier | 996337b9-4571-486e-9d9c-b67cd3f22d81 |
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| Record created | 2010-09-27 |
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| Record modified | 2020-04-17 |
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