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




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BMC Biotechnology

, 13:41

Protein and enzyme technology

Abstract

BackgroundThe 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.

ResultsThe 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.

ConclusionsWe 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.

KeywordsStyrene-maleic acid Copolymer Amphipols Membrane proteins Nanoscale model membranes Mitochondria Lipodisqs® AbbreviationsSMAStyrene maleic acid

amphipolsAmphipathic polymers

DMPC1,2-Dimyristoyl-sn-phosphocholine

POPC1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine

POPE1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine

tetraoleoyl CL1’,3’-bis1,2-dioleoyl-sn-glycero-3-phospho-sn-glycerol

IMInner membrane

∆ψmTransmembrane electric potential

TMRMTetramethyl rhodamine methyl ester

DDMN-dodecyl-β-D-maltoside

BN-PAGEBlue native polyacrylamide gel electrophoresis

TEMTransmission electron microscopy

SECSize exclusion chromatography

KCNPotassium cyanide.

Electronic supplementary materialThe online version of this article doi:10.1186-1472-6750-13-41 contains supplementary material, which is available to authorized users.

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