For successful incorporation of purified MPs ( Fig. This pathway allows the purification step to be bypassed and accelerates the entire process of nanodisc self-assembly, which is sometimes critically important for preservation of the native and functional form of the target MP (illustrated in Fig. If the MP under investigation is unstable to purification in detergent, or a library of all MPs in a specific tissue is desired, incorporation into nanodiscs can be achieved through direct solubilization of cell membranes by using detergents 5 or membrane-active polymers 6, 7 ( Fig. This simple approach provides control over the composition and homogeneity of the resultant assembly and can be optimized to maximize the yield of MP incorporation. The molar stoichiometric ratio of lipid to scaffold protein is important for optimized yield 2, 3, whereas the size and ratio of scaffold protein to MP may be selected to favor incorporation of predominantly monomeric (in which there is a large excess of scaffold protein and lipid) or oligomeric target into nanodiscs 3, 4. MPs of many types and topologies have successfully been self-assembled into nanodiscs, by starting from a detergent-solubilized mixture including all components: target, lipid mixture of choice and membrane scaffold proteins ( Fig. The resultant discoidal bilayers can be made homogeneous and monodisperse and can be obtained with high yield 2, 3.Ī large body of published work has used nanodiscs as a vehicle to incorporate recalcitrant MP targets into the bilayer to preserve MP structure and activity. The size of nanodiscs is determined by the length of the membrane scaffold protein and the stoichiometry of the lipids used in the self-assembly process. Nanodiscs are discoidal lipid bilayers of 8–16 nm in diameter, which are stabilized and rendered soluble in aqueous solutions by two encircling amphipathic helical protein belts, termed membrane scaffold proteins 1, 2. By providing such a native membrane environment, nanodiscs have proven to be an invaluable tool for revealing the structure and function of isolated MPs as well as their complexes with other proteins and lipids. For many systems and projects, a planar bilayer model system of ∼10 nm in diameter would be ideal, providing space for one or more MPs and allowing access to both sides for the assay of signaling events. Ideally, an MP structural study should elucidate not only the conformations and protein-protein interactions in supramolecular complexes but also their topology in the lipid bilayer and the details of the protein-lipid interface, which often are highly specific with respect to lipid composition. Thus, the structural biology of MPs occupies a central place in current biophysics, biochemistry and cell biology investigations. Understanding the molecular mechanisms of MP action is impossible without detailed knowledge of their structure and how they interact with other proteins, nucleic acids and lipids.
Despite these crucial roles, MPs have been notoriously difficult to work with because they often display altered or loss of activity and function outside of a phospholipid environment. Because of their role in the regulation of vital cellular functions, MPs are the target of the majority of currently marketed therapeutics. They are the key conduit for communication between cells, conduct the vital transformations that produce energy, provide channels for the transport of molecules between the inside and outside of cells and intracellular compartments and are the workhorses for a plethora of enzyme-catalyzed metabolic transformations. Membrane proteins (MPs) are central to life processes.
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