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.

The ADP/ATP Carrier (AAC) is the most abundant transporter of the mitochondrial inner membrane. The central role that this transporter plays in cellular energy production highlights the importance of understanding its structure, function, and the basis of its pathologies. As a means of preparing proteoliposomes for the study of membrane proteins, several groups have explored the use of cell-free translation systems to facilitate membrane protein integration directly into preformed unilamellar vesicles without the use of surfactants. Using AAC as a model, we report for the first time the detergent-free reconstitution of a mitochondrial inner membrane protein into liposomes using a wheat germ-based in vitro translation system. Using a host of independent approaches, we demonstrate the efficient integration of AAC into vesicles with an inner membrane-mimetic lipid composition and, more importantly, that the integrated AAC is functionally active in transport. By adding liposomes at different stages of the translation reaction, we show that this direct integration is obligatorily cotranslational, and by synthesizing stable ribosome-bound nascent chain intermediates, we show that the nascent AAC polypeptide interacts with lipid vesicles while ribosome-bound. Finally, we show that the presence of the phospholipid cardiolipin in the liposomes specifically enhances AAC translation rate as well as the efficiency of vesicle association and integration. In light of these results, the possible mechanisms of liposome-assisted membrane protein integration during cell-free translation are discussed with respect to the mode of integration and the role of specific lipids.

this work, we demonstrate ligand-targeted delivery with a novel magnetic microsphere by conjugating the drug carriers with a folic acid ligand that preferentially binds to HeLa cells overexpressing folic acid receptors. The microspheres used in this study are produced in-house and contain magnetite nanoparticles (~10 nm) distributed uniformly throughout an aminefunctionalized silicone matrix. The sphere diameter is scalable from 0.5 to 2.0 microns, and the concentration of magnetic nanoparticles can be varied up to 50% wt. The silicon matrix of this carrier facilitates compatibility with lipophilic drugs, the high magnetic content allows the potential for magnetically-stimulated drug release, and an abundance of primary amines within the matrix enables surface functionalization with a variety of ligands. Microspheres in this study were conjugated with folic acid using an EDAC reaction and tagged with a fluorophore. The spheres were incubated with HeLa cells, which overexpress folate-binding protein, and the degree of binding after 30 minutes was analyzed with fluorescence microscopy. We show a fivefold increase in bound spheres per cell relative to a control sphere without folic acid, indicating a high degree of specific binding. The preferential binding of ligand-conjugated magnetic microspheres gives insight into the utility of these drug carriers for targeted drug delivery studies.