Distribution, lateral mobility and function of membrane proteins incorporated into giant unilamellar vesicles |
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| Authors: | Doeven Mark K Folgering Joost H A Krasnikov Victor Geertsma Eric R van den Bogaart Geert Poolman Bert |
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| Affiliation: | Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute and Materials Science Centre, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands. |
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| Abstract: | GUVs have been widely used for studies on lipid mobility, membrane dynamics and lipid domain (raft) formation, using single molecule techniques like fluorescence correlation spectroscopy. Reports on membrane protein dynamics in these types of model membranes are by far less advanced due to the difficulty of incorporating proteins into GUVs in a functional state. We have used sucrose to prevent four distinct membrane protein(s) (complexes) from inactivating during the dehydration step of the GUV-formation process. The amount of sucrose was optimized such that the proteins retained 100% biological activity, and many proteo-GUVs were obtained. Although GUVs could be formed by hydration of lipid mixtures composed of neutral and anionic lipids, an alternate current electric field was required for GUV formation from neutral lipids. Distribution, lateral mobility, and function of an ATP-binding cassette transport system, an ion-linked transporter, and a mechanosensitive channel in GUVs were determined by confocal imaging, fluorescence correlation spectroscopy, patch-clamp measurements, and biochemical techniques. In addition, we show that sucrose slows down the lateral mobility of fluorescent lipid analogs, possibly due to hydrogen-bonding with the lipid headgroups, leading to larger complexes with reduced mobility. |
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| Keywords: | GUV, giant unilamellar vesicle LUV, large unilamellar vesicle ABC, ATP-binding cassette AC, alternating current DDM, n-dodecyl-β- smallcaps" >d-maltoside DiO, 3,3′-dioctadecyloxacarbocyanine perchlorate (DiOC18(3)) DOPC, 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine DOPE, L-α-dioleoyl phosphatidylethanolamine DOPG, 1,2-dioleolyl-sn-glycerol-3-[phospho-rac-(1-glycerol)] DOPS, 1,2-dioleoyl-sn-glycero-3-phosphatidylserine FCS, fluorescence correlation spectroscopy NBD C6-HPC, , 2-(6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl-1-hexadecanoyl phosphocholine ITO, indium tin oxide and CSSA, , cross-sectional surface area |
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