Circularized nandiscs (cNDs) exhibit superb monodispersity and have the potential to transform functional and structural studies of membrane proteins. In particular, cNDs can stabilize large patches of lipid bilayers for the reconstitution of complex membrane biochemical reactions, enabling the capture of crucial intermediates involved in synaptic transmission and viral entry. However, previous methods for building cNDs require multiple steps and suffer from low yields. We herein introduce a simple, one-step approach to ease the construction of cNDs using the SpyCatcher-SpyTag technology.
Fig: Circularization of MSPs via the SpyCatcher-SpyTag: a The SpyCatcher (sapphire blue) and SpyTag (navy blue) were appended to the N- and C-termini of MSPs for spontaneous circularization of MSPs in cells (upper). Illustration of the circularized nanodiscs by the SpyCatcher-SpyTag (bottom). b Representative SDS-PAGE of circularized (WT) and non-circularized (EQ) MSPs. n = 4 independent experiments. c Quantification of the unreacted peptide in the WT and EQ mutant of spMSP1D1 using mass spectrometry. Data are shown as mean ± s.d., n = 2 independent experiments. Source Data are available as a source data file.
This approach increases the yield of cNDs by over 10-fold and is able to rapidly generates cNDs with diameters ranging from 11 to over 100 nm. We demonstrate the utility of these cNDs for mechanistic interrogations of vesicle fusion and protein-lipid interactions that are unattainable using small nanodiscs. Together, the remarkable performance of SpyCatcher-SpyTag in nanodisc circularization paves the way for the use of cNDs in membrane biochemistry and structural biology.
Zhang, S., Ren, Q., Novick, S.J. et al. One-step construction of circularized nanodiscs using SpyCatcher-SpyTag. Nat Commun 12,5451 (2021). https://doi.org/10.1038/s41467-021-25737-7