Constructing artificial membrane structures on graphene to resolve membrane protein conformational dynamics

Abstract text (incl. figure legends and references)

Mechanistic understanding of membrane-based processes requires detailed insights of the membrane environment and its impact on protein structure and function. Membrane properties such as fluidity, curvature, and lipid phase segregation have tremendous influence to membrane protein structure. To explore the conformational spectra of membrane protein on variable membrane structures, we tether liposomes as membrane curvature models and fuse them into planar lipid bilayer on graphene-coated substrate. For this purpose, ultra small unilamellar vesicles (USUVs) are prepared with a mean hydrodynamic diameter of 40±15nm. Using biotin-streptavidin interaction, stable immobilization of the USUVs on graphene-supported lipid monolayer (GSLM) is confirmed by negative staining electron microscopy (EM). The USUVs can thus be used to integrate individual ABC transporter MsbA per liposome on graphene-coated EM grid to determine the correlation of protein structure with membrane curvature. By fusing the USUVs on GSLM on glass substrate, fluorescence recovery after photobleaching reveals a fluidic membrane structure. Based on these artificial membrane structures on graphene, we aim to combine single molecule fluorescence microscopy and cryo-EM to correlatively investigate the time-resolved conformational dynamics of membrane protein in controlled membrane environment.