Journal
EUROPEAN BIOPHYSICS JOURNAL WITH BIOPHYSICS LETTERS
Volume 44, Issue 7, Pages 503-512Publisher
SPRINGER
DOI: 10.1007/s00249-015-1032-y
Keywords
Gel-shift assay; Membrane enzymes; Outer membrane phospholipase A; Proteoliposomes; Protein refolding
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Funding
- Deutsche Forschungsgemeinschaft (DFG) through International Research Training Group IRTG 1830
- Research Initiative BioComp
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Bilayer mechanical properties are not only of crucial importance to the mechanism of action of mechanosensation in lipid membranes but also affect preparative laboratory tasks such as membrane-protein refolding. We report this for coupled refolding and bilayer insertion of outer membrane phospholipase A (OmpLA), an integral membrane enzyme that catalyses the hydrolytic cleavage of glycerophospholipids. OmpLA can be refolded into a variety of detergent micelles and unilamellar vesicles composed of short-chain phospholipids but, in the absence of chemical or molecular chaperones, not into thicker membranes. Controlled modulation of bilayer mechanical properties by judicious use of subsolubilising concentrations of detergents induces monolayer curvature strain, acyl chain fluidisation, membrane thinning, and transient aqueous bilayer defects. This enables quantitative and functional refolding of OmpLA even into bilayer membranes composed of long-chain phospholipids to yield enzymatically active proteoliposomes without requiring membrane solubilisation.
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