Reconstitution of membrane proteins into giant unilamellar vesicles via peptide-induced fusion.
In this work, we present a protocol to reconstitute membrane proteins into giant unilamellar vesicles (GUV) via peptide-induced fusion. In principle, GUV provide a well-defined lipid matrix, resembling a close-to-native state for biophysical studies, including optical microspectroscopy, of transmembrane proteins at the molecular level. Furthermore, reconstitution in this manner would also eliminate potential artifacts arising from secondary interactions of proteins, when reconstituted in planar membranes supported on solid surfaces. However, assembly procedures of GUV preclude direct reconstitution. Here, for the first time, a method is described that allows the controlled incorporation of membrane proteins into GUV. We demonstrate that large unilamellar vesicles (LUV, diameter 0.1 microm), to which the small fusogenic peptide WAE has been covalently attached, readily fuse with GUV, as revealed by monitoring lipid and contents mixing by fluorescence microscopy. To monitor contents mixing, a new fluorescence-based enzymatic assay was devised. Fusion does not introduce changes in the membrane morphology, as shown by fluorescence correlation spectroscopy. Analysis of fluorescence confocal imaging intensity revealed that approximately 6 to 10 LUV fused per microm(2) of GUV surface. As a model protein, bacteriorhodopsin (BR) was reconstituted into GUV, using LUV into which BR was incorporated via detergent dialysis. BR did not affect GUV-LUV fusion and the protein was stably inserted into the GUV and functionally active. Fluorescence correlation spectroscopy experiments show that BR inserted into GUV undergoes unrestricted Brownian motion with a diffusion coefficient of 1.2 microm(2)/s. The current procedure offers new opportunities to address issues related to membrane-protein structure and dynamics in a close-to-native state.
Kahya
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Ultrafast Laser and Spectroscopy Laboratory, Optical Sciences, Materials Science Centre, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands. kahya@chem.rug.nl
Pécheur
E I
EI
de Boeij
W P
WP
Wiersma
D A
DA
Hoekstra
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D
D001436:Bacteriorhodopsins / Q000378:metabolism; D004058:Diffusion; D008081:Liposomes / Q000737:chemistry* / Q000378:metabolism*; D008561:Membrane Fusion / Q000187:drug effects*; D008563:Membrane Lipids / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008858:Microscopy, Phase-Contrast; D010455:Peptides / Q000494:pharmacology*; D010713:Phosphatidylcholines / Q000378:metabolism; D017494:Proton Pumps / Q000378:metabolism
D008081:Liposomes; D008563:Membrane Lipids; D008565:Membrane Proteins; D010455:Peptides; D010713:Phosphatidylcholines; D017494:Proton Pumps; D001436:Bacteriorhodopsins; C017251:1,2-oleoylphosphatidylcholine