Abstract
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. 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.
Highlights
Circularized nandiscs exhibit superb monodispersity and have the potential to transform functional and structural studies of membrane proteins
Despite the power to reconstitute crucial reactions involved in synaptic transmission and viral entry, the sophisticated procedures and low yields for building these Circularized nandiscs (cNDs) limit their broad application in membrane biochemistry
We posit that this conjugation method would increase the yield of cNDs, since the spontaneous in vivo circularization of MSP could improve the protein solubility and stability by protecting its hydrophobic face from aggregation and degradation
Summary
Circularized nandiscs (cNDs) exhibit superb monodispersity and have the potential to transform functional and structural studies of membrane proteins. This new method exhibits nearly diffusion-limited kinetics and has enabled circularization of several proteins with enhanced stabilities[15,16,17,18,19] Inspired by these studies, we simplify the production of cNDs using SpyCatcher-SpyTag to circularize MSPs in cells, thereby by passing the in vitro protein refolding, sortase-mediated ligation, and repurification steps as required in the previous study. We simplify the production of cNDs using SpyCatcher-SpyTag to circularize MSPs in cells, thereby by passing the in vitro protein refolding, sortase-mediated ligation, and repurification steps as required in the previous study This approach greatly enhances the solubility and yield of circularized large MSPs to the same level as traditional small MSPs. we could readily build cNDs with diameters from 11 to over 100 nm, which allows us to reconstitute protein-lipid interactions and membrane fusion intermediates that are unattainable using small nanodiscs. These circularized MSPs will significantly promote the use of cNDs for biochemical and structural studies of membrane proteins
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