title: Paramagnetic proteoliposomes containing a pure, native, and oriented seven-transmembrane segment protein, CCR5.
abstract: Seven-transmembrane segment, G protein-coupled receptors play central roles in a wide range of biological processes, but their characterization has been hindered by the difficulty of obtaining homogeneous preparations of native protein. We have created paramagnetic proteoliposomes containing pure and oriented CCR5, a seven-transmembrane segment protein that serves as the principal coreceptor for human immunodeficiency virus (HIV-1). The CCR5 proteoliposomes bind the HIV-1 gp120 envelope glycoprotein and conformation-dependent antibodies against CCR5. The binding of gp120 was enhanced by a soluble form of the other HIV-1 receptor, CD4, but did not require additional cellular proteins. Paramagnetic proteoliposomes are uniform in size, stable in a broad range of salt concentrations and pH, and can be used in FACS and competition assays typically applied to cells. Integral membrane proteins can be inserted in either orientation into the liposomal membrane. The magnetic properties of these proteoliposomes facilitate rapid buffer exchange useful in multiple applications. As an example, the CCR5-proteoliposomes were used to select CCR5-specific antibodies from a recombinant phage display library. Thus, paramagnetic proteoliposomes should be useful tools in the analysis of membrane protein interactions with extracellular and intracellular ligands, particularly in establishing screens for inhibitors.
authors.affiliation: Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, 44 Binney St., Boston, MA 02115, USA.
authors.forename: T
authors.initials: T
authors.lastname: Mirzabekov
authors.forename: H
authors.initials: H
authors.lastname: Kontos
authors.forename: M
authors.initials: M
authors.lastname: Farzan
authors.forename: W
authors.initials: W
authors.lastname: Marasco
authors.forename: J
authors.initials: J
authors.lastname: Sodroski
mesh_terms: D000911:Antibodies, Monoclonal / Q000378:metabolism; D015704:CD4 Antigens / Q000737:chemistry / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004306:Dose-Response Relationship, Immunologic; D004591:Electrophoresis, Polyacrylamide Gel; D005434:Flow Cytometry; D015699:HIV Envelope Protein gp120 / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D008051:Lipid Bilayers / Q000737:chemistry; D008280:Magnetics; D018613:Microscopy, Confocal; D008954:Models, Biological; D019151:Peptide Library; D011485:Protein Binding; D011487:Protein Conformation; D011510:Proteolipids / Q000737:chemistry*; D019713:Receptors, CCR5 / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D015704:CD4 Antigens; D015699:HIV Envelope Protein gp120; D008024:Ligands; D008051:Lipid Bilayers; D019151:Peptide Library; D011510:Proteolipids; D019713:Receptors, CCR5; C020485:proteoliposomes