title: Preparation of semisynthetic lipoproteins with fluorescent cholesterol anchor and their introduction to the cell membrane with minimal disruption of the membrane.
abstract: The exogenous introduction of fluorescent lipoproteins into cell membranes is a method for visualizing the cellular traffic of membrane associated proteins, and also for altering the cell surface in a controlled manner. In order to achieve the cell membrane anchoring of proteins and their subsequent fluorescence based detection, a cholesterol derivative was designed. The headgroup of the novel cholesterol anchor contains a fluorescent reporter and a thiol reactive maleimide for protein conjugation. Protein conjugation was demonstrated by the addition of a green fluorescent maleimido anchor to the C-terminus of a Cys extended red fluorescent protein, mCherry. The resulting dual fluorescent cholesteryl lipoprotein was successfully separated from the micellar associates of the surplus fluorescent lipid anchor without denaturing the protein, and the lipoprotein containing only the covalently linked, stoichiometric fluorescent lipid was efficiently delivered to the plasma membrane of live cells. It was demonstrated that the membrane fluorescence could be directly assigned to the protein-anchor conjugate, because no excess of fluorescent lipid species were present during the imaging experiment and the protein and anchor fluorescence colocalized in the cell membrane. Molecular dynamics simulations and subsequent trajectory analysis suggest also the spontaneous and stable membrane association of the cholesterol anchor. Thus, the method could be beneficially applied for studying membrane associated proteins and for preparing mimetics of glycosylphosphatidylinositol (GPI)-anchored proteins to target cholesterol-rich membrane microdomains.
authors.affiliation: Laboratory of Chemical Biology and §Laboratory of Conformational Diseases, Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of Sciences , Temesvári krt. 62., 6726 Szeged, Hungary.
authors.forename: Balázs
authors.initials: B
authors.lastname: Schäfer
authors.forename: Erika
authors.initials: E
authors.lastname: Orbán
authors.forename: Attila
authors.initials: A
authors.lastname: Borics
authors.forename: Krisztina
authors.initials: K
authors.lastname: Huszár
authors.forename: Antal
authors.initials: A
authors.lastname: Nyeste
authors.forename: Ervin
authors.initials: E
authors.lastname: Welker
authors.forename: Csaba
authors.initials: C
authors.lastname: Tömböly
mesh_terms: D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002784:Cholesterol / Q000031:analogs & derivatives* / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008074:Lipoproteins / Q000737:chemistry* / Q000378:metabolism; D008164:Luminescent Proteins / Q000737:chemistry* / Q000378:metabolism; D000097573:Red Fluorescent Protein
chemical_list: D002784:Cholesterol; D005456:Fluorescent Dyes; D008074:Lipoproteins; D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein