Reconstitution of SNARE proteins into solid-supported lipid bilayer stacks and X-ray structure analysis

摘要

Abstract SNAREs are known as an important family of proteins mediating vesicle fusion. For various biophysical studies, they have been reconstituted into supported single bilayers via proteoliposome adsorption and rupture. In this study we extended this method to the reconstitution of SNAREs into supported multilamellar lipid membranes, i.e. oriented multibilayer stacks, as an ideal model system for X-ray structure analysis (X-ray reflectivity and diffraction). The reconstitution was implemented through a pathway of proteomicelle, proteoliposome and multibilayer. To monitor the structural evolution in each step, we used small-angle X-ray scattering for the proteomicelles and proteoliposomes, followed by X-ray reflectivity and grazing-incidence small-angle scattering for the multibilayers. Results show that SNAREs can be successfully reconstituted into supported multibilayers, with high enough orientational alignment for the application of surface sensitive X-ray characterizations. Based on this protocol, we then investigated the effect of SNAREs on the structure and phase diagram of the lipid membranes. Beyond this application, this reconstitution protocol could also be useful for X-ray analysis of many further membrane proteins. Graphical Abstract Display Omitted Highlights ? A protocol for solvent-free reconstitution of membrane proteins into highly oriented lipid bilayer stacks for x-ray analysis is presented. ? The entire preparation pathway is carefully investigated by SAXS, reflectivity and GISAXS. ? Soluble NSF-attachment protein receptors (SNAREs) are reconstituted into model bilayer stacks. ? Residual detergents are found to largely affect the bilayer structures and its phase behavior.