Reconstitution of Membrane-associated Components of a G-protein Signaling Pathway on Membrane-coated Nanoparticles (Lipobeads).

Academic Article


  • G-protein coupled signaling pathways are organized into multi-protein complexes called signalosomes that are located within and on cellular membranes. We describe the use of silica nanoparticles coated with a unilamellar phospholipid bilayer (lipobeads) to reconstitute the activated photoreceptor G-protein α-subunit (Gtα*) with its cognate effector (phosphodiesterase-6; PDE6) for biochemical and structural studies of the activation mechanism regulating this GPCR signaling pathway. Lipobeads are prepared by resuspending dried-down phospholipid mixtures with monodisperse 70 nm silica particles, followed by extrusion through a 100 nm membrane filter. This uniform and supported liposomal preparation is easily sedimented, permitting the separation of soluble from membrane-associated proteins. Upon loading lipobeads with Gtα* and PDE6, we find that activation of PDE6 catalysis by Gtα* occurs much more efficiently than in the absence of membranes. Chemical cross-linking of membrane-confined proteins allows detection of changes in protein-protein interactions, resulting from G-protein activation of PDE6. The advantages of using lipobeads over partially purified membrane preparations or traditional liposomal preparations are generally applicable to the study of other membrane-confined signal transduction pathways.
  • Authors

  • Irwin, Michael J
  • Wang, Xin
  • Cote, Rick
  • Publication Date

  • January 20, 2022
  • Published In

  • Bio Protoc  Journal
  • Keywords

  • G-protein
  • Liposome
  • Nanoparticle
  • Peripheral membrane protein
  • Protein-protein interactions
  • Signal transduction
  • Signalosome
  • Digital Object Identifier (doi)

    Start Page

  • e4303
  • Volume

  • 12
  • Issue

  • 2