A detergent-free strategy for the reconstitution of active enzyme complexes from native biological membranes into nanoscale discs

BMC Biotechnology

Ashley R Long²  Catherine C O’Brien²  Ketan Malhotra²  Christine T Schwall²  Arlene D Albert²  Anthony Watts¹  Nathan N Alder² 

[1] Department of Biochemistry, Biomembrane Structure Unit, University of Oxford, Oxford, OX1 3QU, UK

[2] Department of Molecular and Cell Biology, University of Connecticut, 91 North Eagleville Rd, Storrs, Connecticut 06269, USA

关键词: Lipodisqs®;    Mitochondria;    Nanoscale model membranes;    Membrane proteins;    Amphipols;    Copolymer;    Styrene-maleic acid;   

Others  :  1123173
DOI  :  10.1186/1472-6750-13-41

 received in 2013-01-15, accepted in 2013-05-07,  发布年份 2013

【 摘 要 】

Background

The reconstitution of membrane proteins and complexes into nanoscale lipid bilayer structures has contributed significantly to biochemical and biophysical analyses. Current methods for performing such reconstitutions entail an initial detergent-mediated step to solubilize and isolate membrane proteins. Exposure to detergents, however, can destabilize many membrane proteins and result in a loss of function. Amphipathic copolymers have recently been used to stabilize membrane proteins and complexes following suitable detergent extraction. However, the ability of these copolymers to extract proteins directly from native lipid bilayers for subsequent reconstitution and characterization has not been explored.

Results

The styrene-maleic acid (SMA) copolymer effectively solubilized membranes of isolated mitochondria and extracted protein complexes. Membrane complexes were reconstituted into polymer-bound nanoscale discs along with endogenous lipids. Using respiratory Complex IV as a model, these particles were shown to maintain the enzymatic activity of multicomponent electron transporting complexes.

Conclusions

We report a novel process for reconstituting fully operational protein complexes directly from cellular membranes into nanoscale lipid bilayers using the SMA copolymer. This facile, single-step strategy obviates the requirement for detergents and yields membrane complexes suitable for structural and functional studies.

【 授权许可】

   
2013 Long et al.; licensee BioMed Central Ltd.

【 图 表 】

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