Journal
CHEMBIOCHEM
Volume 12, Issue 18, Pages 2856-2862Publisher
WILEY-BLACKWELL
DOI: 10.1002/cbic.201100537
Keywords
fluorescence; generalized polarization; giant protein vesicles; membrane proteins; reconstitution
Funding
- European Commission [NMP4-CT-2006-033234]
- Danish National Advanced Technology Foundation [023-2007-1]
- Environment & Water Industry Development Council of Singapore (EWI) [MEWR 651/06/169]
- Singapore National Research Foundation [NRF-CRP4-2008-02, 0804-IRIS-02]
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This paper describes a method to create giant protein vesicles (GPVs) of >= 10 mu m by solvent-driven fusion of large vesicles (0.10.2 mu m) with reconstituted membrane proteins. We found that formation of GPVs proceeded from rotational mixing of protein-reconstituted large unilamellar vesicles (LUVs) with a lipid-containing solvent phase. We made GPVs by using n-decane and squalene as solvents, and applied generalized polarization (GP) imaging to monitor the polarity around the protein transmembrane region of aquaporins labeled with the polarity-sensitive probe Badan. Specifically, we created GPVs of spinach SoPIP2;1 and E. coli AqpZ aquaporins. Our findings show that hydrophobic interactions within the bilayer of formed GPVs are influenced not only by the solvent partitioning propensity, but also by lipid composition and membrane protein isoform.
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