Structural and functional analysis of cell-free expressed CCR5, a coreceptor required for HIV infection; and establishment of a new detergent free reconstitution method.

• Antragsteller: Dr. Christian Klammt
• Fachliche Zuordnung: Biochemie
• Förderung: Förderung von 2007 bis 2008
• Projektkennung: Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 60843850

G-protein coupled receptors (GPCRs) represent by far the largest class of targets for modern drugs. The structural analysis of these receptors has so far almost been inconceivable because of an insufficient sample preparation in conventional cellular expression systems. I propose to overcome this problem by using cell-free (CF) expression. It has recently been demonstrated that CF systems provide an emerging alternative tool for the high level production of integral membrane proteins (IMPs) including human GPCRs. I will analyze the inflammatory CC chemokines receptor 5 (CCR5) for high level CF expression and structural analysis. CCR5 is playing a predominant role in the human immunodeficiency virus (HIV) infection as HIV requires CCR5 as coreceptor for cell-entry into leukocytes. Preliminary data shows that CF expression will provide sufficient amounts of this pharmacologically interesting receptor for crystallization experiments and functional analysis. Recently, Senyon Choe’s group at the Salk Institute discovered a membrane protein, Mistic, which, when fused N-terminally to various IMPs, is able to guide its cargo protein into the E. coli membrane for autonomous insertion and folding. I will further evaluate a new mode for CF expression of IMPs on the basis of Mistic guided membrane insertion. This new approach of CF expression will be tested by fusing Mistic to CCR5 and by monitoring lipid reconstitution in the absence of any detergent. Success in this study will provide a new platform for bilayer 2 D and 3D crystallization and functional analysis of CCR5.

• DFG-Verfahren: Forschungsstipendien
• Internationaler Bezug: USA
• Gastgeber: Professor Senyon Choe, Ph.D.