Fig. 2: Developing nanodisc-ID to characterize protein–lipid interactions.

A Illustration of nanodisc-ID. Top, PL enzymes were fused to MSPs for developing the nanodisc-ID approach. Bottom, binding of pMPs to nanodiscs results in biotin labeling by PL enzymes. B The indicated MSPs were fused with TurboID and were then used to form nanodiscs containing PI lipids. PL efficiencies of syt1 by these nanodiscs were quantified. Data are shown as mean ± s.d., n = 3 independent experiments. C Representative size-exclusion chromatography (SEC) profile of TurboID-18A NDs containing PI lipids. D Representative negative stain image of the fractions corresponding to NDs from SEC in C. Scale bar: 20 nm. E Quantification of TurboID-18A ND diameters containing PI lipids from negative stain EM (n = 57). F The cytosolic domain of synaptotagmin-1 (syt1) was labeled with 5-IAF and incubated with TurboID-18A nanodiscs containing 10% of the indicated lipids and 90% of PC, followed by PL reactions. Samples, with or without the addition of SA, were subjected to SDS-PAGE and in-gel fluorescent imaging. PC 1,2-dioleoyl-sn-glycero-3-phosphocholine, PS 1,2-dioleoyl-sn-glycero-3-phospho-l-serine, PE 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, PI brain L-α-phosphatidylinositol-4,5-bisphosphate, CL E.coli cardiolipin.