title: A detergent-free strategy for the reconstitution of active enzyme complexes from native biological membranes into nanoscale discs.
abstract: 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.
authors.affiliation: Department of Molecular and Cell Biology, University of Connecticut, 91 North Eagleville Rd, Storrs, Connecticut 06269, USA.
authors.forename: Ashley R
authors.initials: AR
authors.lastname: Long
authors.forename: Catherine C
authors.initials: CC
authors.lastname: O'Brien
authors.forename: Ketan
authors.initials: K
authors.lastname: Malhotra
authors.forename: Christine T
authors.initials: CT
authors.lastname: Schwall
authors.forename: Arlene D
authors.initials: AD
authors.lastname: Albert
authors.forename: Anthony
authors.initials: A
authors.lastname: Watts
authors.forename: Nathan N
authors.initials: NN
authors.lastname: Alder
mesh_terms: D001709:Biotechnology / Q000379:methods*; D008298:Maleates / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D051336:Mitochondrial Membranes / Q000201:enzymology*; D008954:Models, Biological; D009097:Multienzyme Complexes / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D053758:Nanoparticles / Q000737:chemistry* / Q000378:metabolism; D011137:Polystyrenes / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D008298:Maleates; D008565:Membrane Proteins; D009097:Multienzyme Complexes; D011137:Polystyrenes; D029701:Saccharomyces cerevisiae Proteins; C024074:styrene-maleic acid polymer