Paramagnetic proteoliposomes containing a pure, native, and oriented seven-transmembrane segment protein, CCR5.

Important note: Information in this article was accurate in 2000. The state of the art may have changed since the publication date.

Paramagnetic proteoliposomes containing a pure, native, and oriented seven-transmembrane segment protein, CCR5.

Nat Biotechnol. 2000 Jun;18(6):649-54. Unique Identifier : AIDSLINE MED/20296931
Mirzabekov T; Kontos H; Farzan M; Marasco W; Sodroski J; Department of Cancer Immunology and AIDS, Dana-Farber Cancer; Institute, 44 Binney St., Boston, MA 02115, USA.

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.

Keywords: JOURNAL ARTICLE Antibodies, Monoclonal/METABOLISM Antigens, CD4/CHEMISTRY/METABOLISM Cell Line Cell Membrane/CHEMISTRY Dose-Response Relationship, Drug Dose-Response Relationship, Immunologic Electrophoresis, Polyacrylamide Gel Flow Cytometry Human Hydrogen-Ion Concentration HIV Envelope Protein gp120/CHEMISTRY/METABOLISM Ligands Lipid Bilayers/CHEMISTRY Magnetics Microscopy, Confocal Models, Biological Peptide Library Protein Binding Protein Conformation Proteolipids/*CHEMISTRY Receptors, CCR5/*CHEMISTRY/IMMUNOLOGY/METABOLISM Support, Non-U.S. Gov't Support, U.S. Gov't, P.H.S.

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