title: A biosensor-based approach toward purification and crystallization of G protein-coupled receptors.
abstract: Biacore technology was used to develop an affinity purification method and screen cocrystallization conditions for the chemokine receptor CCR5. We characterized the binding of nine HIV gp120 variants and identified a truncated construct (YU2DV1V2) that bound CCR5 independent of CD4. This construct was used in an affinity purification step to improve the activity of detergent-solubilized receptor by approximately 300%. The biosensor was also used to screen receptor binding activity automatically under 50 different crystallization conditions. We found that high-molecular-weight polyethylene glycols (PEGs 4,000 and 8,000 Da) most often stabilized the receptor and improved complex formation with potential cocrystallization partners such as conformationally sensitive monoclonal antibodies and gp120. Our results show how biosensors can provide unique insights into receptor purification methods and reveal the effects of crystallization conditions on complex formation. Importantly, these methods can be readily applied to other systems.
authors.affiliation: Center for Biomolecular Interaction Analysis, School of Medicine, University of Utah, Salt Lake City, 84132, USA.
authors.forename: Iva
authors.initials: I
authors.lastname: Navratilova
authors.forename: Marie
authors.initials: M
authors.lastname: Pancera
authors.forename: Richard T
authors.initials: RT
authors.lastname: Wyatt
authors.forename: David G
authors.initials: DG
authors.lastname: Myszka
mesh_terms: D000911:Antibodies, Monoclonal / Q000378:metabolism; D015374:Biosensing Techniques / Q000379:methods*; D002846:Chromatography, Affinity; D003460:Crystallization; D015699:HIV Envelope Protein gp120 / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D019713:Receptors, CCR5 / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D012995:Solubility; D013997:Time Factors
chemical_list: D000911:Antibodies, Monoclonal; D015699:HIV Envelope Protein gp120; D019713:Receptors, CCR5