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PMID: 15574707 Published · ppublish English Comparative Study Journal Article Research Support, Non-U.S. Gov't

Distribution, lateral mobility and function of membrane proteins incorporated into giant unilamellar vesicles.

Biophysical journal ·Vol. 88 ·No. 2 ·2005-02-00 ·Pages 1134-42

Doeven MK, Folgering JH, Krasnikov V, Geertsma ER, van den Bogaart G, Poolman B

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.

MeSH Terms
ATP-Binding Cassette Transporters/analysis,chemistry,ultrastructure Binding Sites Desiccation Escherichia coli Proteins/analysis,chemistry,ultrastructure Ion Channels/analysis,chemistry,ultrastructure Lactococcus lactis/chemistry Lipid Bilayers/chemistry Liposomes/chemistry Membrane Microdomains/chemistry,ultrastructure Membrane Transport Proteins/analysis,chemistry,ultrastructure Motion Protein Binding Sucrose/chemistry
Chemicals
ATP-Binding Cassette Transporters Escherichia coli Proteins Ion Channels Lipid Bilayers Liposomes Membrane Transport Proteins MscL protein, E coli Sucrose lactose permease
Authors & Affiliations
6 authors, click to expand affiliations / ORCID
Doeven Mark K
Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute and Materials Science Centre, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
Folgering Joost H A
Krasnikov Victor
Geertsma Eric R
van den Bogaart Geert
Poolman Bert
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Article Info
Journal
Biophysical journal
Abbr.
Biophys J
ISSN
0006-3495
DOI
10.1529/biophysj.104.053413
Published
2005-02-00
Epub
2004-00-01
Pages
1134-42
Language
English
Region
United States
NLM ID
0370626
PMCID
PMC1305118
Subset
IM
External Links
PubMed source DOI: 10.1529/biophysj.104.053413 PMC Full Text View journal details
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