AbstractsBiology & Animal Science

Studies on the functionality of biotinylated rhodopsin with biophysical techniques

by Qun Liu

Institution: Freie Universität Berlin
Degree: PhD
Year: 2003
Record ID: 1099045
Full text PDF: http://edocs.fu-berlin.de/diss/receive/FUDISS_thesis_000000001014


Intermolecular transduction of the light signal in rods is initiated by the interaction of the photoreceptor, rhodopsin, with the G protein transducin (Gt). In recent years, research on the rhodopsin/Gt-system has contributed substantially towards our understanding of G protein-coupled signal transduction. Aim of the proposed project is to explore the mechanism of receptor activation and the detailed investigation of the interface between receptor and G protein involved in nucleotide exchange catalysis by biophysical techniques. In the previous work in the Institut für Medizinische Physik und Biophysik it has been studied on how transitory states of the catalytic process, including GDP release, formation of the "empty site"-complex, and GTP-uptake can be regulated in detail. By applying evanescent field techniques like surface plasmon resonance (SPR / BIAcore analysis) it was possible to monitor the interaction between Gt and rhodopsin and the activation of Gt. For these measurements solubilized rhodopsin from rod diskmembranes was chemically modified and immobilized onto the surface of a sensor chip (Bieri et al., 1999). In this thesis recombinant techniques were applied to produce rhodopsins, which are further biotinylated enzymatically at a single, unique site. We intend to purify the rhodopsin via this biotin-tag and to immobilize the protein through a NeutrAvidin linker in a specific orientation onto a sensor surface. This would then allow to measure the interactions between Gt and recombinant rhodopsin, and perhaps opens the door to chip based assays of interactions between G-proteins and G-protein coupled receptors in general. In our approach we translationally fused a 13 amino acid acceptor peptide, which is recognized by the E. coli biotin ligase (BirA protein) to the N-terminus of rhodopsin. Attempts have been made to biotinylate the fused peptide substrate enzymatically in vitro by using recombinant biotin ligase. Western blot techniques were used to detect successful transfer of biotin to the recombinant receptor. After purification, the biotinylated rhodopsin was characterized spectroscopically and its capability to catalyze nucleotide exchange in transducin was determined with established fluorescence assays. Finally, the activation of Gt was monitored using evanescent field techniques (SPR/resonant mirror), Rhodopsin was successfully reconstituted into supported lipid bilayers (SLB) and the coupling reactions with transducin closely resemble those of the native system, indicating that the functionality of rhodopsin was restored in the SLB. Immobilized rhodopsin can be regenerated with 11-cis-retinal, yielding flash-induced activities similar to native rhodopsin. The reconstituted rhodopsin-Gt system was remarkably stable, and repeated activation/deactivation cycles could be monitored readily. This approach may be extended to rhodopsin mutants and the interaction of rhodopsin with other proteins of the visual cascade. Also this approach may be applicable to other G protein coupled…