title: Synthesis and structural characterization of a mimetic membrane-anchored prion protein.
abstract: During pathogenesis of transmissible spongiform encephalopathies (TSEs) an abnormal form (PrP(Sc)) of the host encoded prion protein (PrP(C)) accumulates in insoluble fibrils and plaques. The two forms of PrP appear to have identical covalent structures, but differ in secondary and tertiary structure. Both PrP(C) and PrP(Sc) have glycosylphospatidylinositol (GPI) anchors through which the protein is tethered to cell membranes. Membrane attachment has been suggested to play a role in the conversion of PrP(C) to PrP(Sc), but the majority of in vitro studies of the function, structure, folding and stability of PrP use recombinant protein lacking the GPI anchor. In order to study the effects of membranes on the structure of PrP, we synthesized a GPI anchor mimetic (GPIm), which we have covalently coupled to a genetically engineered cysteine residue at the C-terminus of recombinant PrP. The lipid anchor places the protein at the same distance from the membrane as does the naturally occurring GPI anchor. We demonstrate that PrP coupled to GPIm (PrP-GPIm) inserts into model lipid membranes and that structural information can be obtained from this membrane-anchored PrP. We show that the structure of PrP-GPIm reconstituted in phosphatidylcholine and raft membranes resembles that of PrP, without a GPI anchor, in solution. The results provide experimental evidence in support of previous suggestions that NMR structures of soluble, anchor-free forms of PrP represent the structure of cellular, membrane-anchored PrP. The availability of a lipid-anchored construct of PrP provides a unique model to investigate the effects of different lipid environments on the structure and conversion mechanisms of PrP.
authors.affiliation: Department of Biological Sciences, University of Warwick, Coventry, UK.
authors.forename: Matthew R
authors.initials: MR
authors.lastname: Hicks
authors.forename: Andrew C
authors.initials: AC
authors.lastname: Gill
authors.forename: Imanpreet K
authors.initials: IK
authors.lastname: Bath
authors.forename: Atvinder K
authors.initials: AK
authors.lastname: Rullay
authors.forename: Ian D
authors.initials: ID
authors.lastname: Sylvester
authors.forename: David H
authors.initials: DH
authors.lastname: Crout
authors.forename: Teresa J T
authors.initials: TJ
authors.lastname: Pinheiro
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D017261:Glycosylphosphatidylinositols / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D008081:Liposomes / Q000737:chemistry; D021962:Membrane Microdomains; D008565:Membrane Proteins / Q000235:genetics; D008954:Models, Biological; D011328:Prions / Q000138:chemical synthesis* / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary
chemical_list: D017261:Glycosylphosphatidylinositols; D008081:Liposomes; D008565:Membrane Proteins; D011328:Prions