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.
Highlights
The reconstitution of membrane proteins and complexes into nanoscale lipid bilayer structures has contributed significantly to biochemical and biophysical analyses
The respiratory complexes of the inner membrane (IM) generate a redox-coupled proton gradient across the IM, the majority of which is stored as a transmembrane electric potential (Δψm, 150–180 mV; Figure 2A) [28]
The relative Transmembrane electric potential (Δψm) can be assessed by the fluorescent potentiometric probe tetramethyl rhodamine methyl ester (TMRM), whose fluorescence intensity decreases with increasing membrane potential [29,30,31]
Summary
The reconstitution of membrane proteins and complexes into nanoscale lipid bilayer structures has contributed significantly to biochemical and biophysical analyses. Integral membrane proteins are vital to cellular function They represent nearly a third of all gene products and account for roughly half of all current pharmaceutical targets [1,2]. These proteins are embedded in the membrane bilayer, which supports the lipid and protein interactions essential for protein function and structural stability. Native lipid bilayers present specific physical and chemical properties to an integral membrane protein in the interfacial and nonpolar regions and generate an environment that is critical for membrane protein function, stability and folding [3,4,5,6] Such an environment is not well represented in micellar structures. It has long been observed that many integral membrane proteins can denature and lose function once removed from the bilayer by detergent solubilization [6,7,8,9,10,11,12]
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