Figure 4. Engineering custom membranes.

I: During nanodisc assembly, many lipid bilayer characteristics can be custom designed. (A) The MSP will define the nanodisc diameter. (B) The lipid composition can be freely chosen, allowing for incorporation of special lipids. (C) The length of the lipid acyl chain will define the hydrophobic thickness of the membrane. Nanodisc reconstitution with a short-chain lipid stabilized MscS in the shown subconducting state. In contrast, reconstitution with a long-chain lipid preserved MscS in the closed state, shown in the nanodisc at the center. II: The membrane can be modified after nanodisc assembly, which will affect both leaflets. (D) Incubation with empty cyclodextrins (CDs) will extract lipids from the membrane, thus increasing membrane tension. This approach stabilized MscS in the shown desensitized conformation. (E) Incubation of nanodiscs with lipid-loaded CDs will exchange lipids in the nanodiscs. Coincubation with lipid-loaded CDs and lipid vesicles would drive lipid addition to the nanodiscs. III: To modify just one leaflet, the protein is reconstituted into vesicles, the outer leaflet is modified, and amphiphilic copolymers are used to form native nanodiscs. (F-H) YnaI reconstituted into vesicles has a closed conformation (F). Incubation with lysophospholipids induced channel opening (G). Extraction with amphiphilic copolymers generated native nanodiscs preserving YnaI in the shown open conformation (H). (I) CDs catalyze the exchange of the outer leaflet lipids between acceptor proteoliposomes and donor vesicles, generating an asymmetric membrane for the embedded protein. (J) Subsequent solubilization with amphiphilic copolymers should yield native nanodiscs of defined leaflet asymmetry.