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Biophys. J. BioFAST: First Published December 1, 2004. doi:10.1529/biophysj.104.053413
© 2004 by the Biophysical Society.

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MEMBRANES

Distribution, Lateral Mobility and Function of Membrane Proteins Incorporated into Giant Unilamellar Vesicles

Mark Doeven 1, Joost Folgering 1, Victor Krasnikov 1, Eric Geertsma 1, Geert van den Bogaart 1 and Bert Poolman 1*

1 University of Groningen

* To whom correspondence should be addressed. E-mail: b.poolman{at}chem.rug.nl.

Submitted on September 23, 2004
Revised on November 12, 2004
Accepted on 18 November 2004


  Abstract
Giant Unilamellar Vesicles (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 (FCS). Reports on membrane protein dynamics in these type 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 AC electric field was required for GUV formation from neutral lipids. Distribution, lateral mobility and function of an ATP-binding cassette (ABC) transport system, an ion-linked transporter and a mechanosensitive channel in GUVs were determined by confocal imaging, FCS, patch-clamp measurements and biochemical techniques. In addition, we show that sucrose slows down the lateral mobility of fluorescent lipid analogues, possibly due to hydrogen-bonding with the lipid headgroups, leading to larger complexes with reduced mobility.

Key Words: electrophysiology, fluorescence correlation spectroscopy, giant unilamellar vesicles, membrane proteins, membrane reconstitution, single molecule spectroscopy




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Copyright © 2004 by the Biophysical Society.