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Kirchhofer, Axel (2009): Structural and Functional Analysis of RIG-I Like Helicases -: Modulating Spectral Properties of the Green Fluorescent Protein with Nanobodies. Dissertation, LMU München: Faculty of Chemistry and Pharmacy
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Abstract

The cytosolic helicases RIG-I and MDA5 are primary sensors for viral RNA during infection. Although their overall role as key players in the antiviral response and the induced signaling pathways have been elucidated in great detail over the past years, a structural and functional understanding of virus recognition by these sensors is missing. On the basis of an X-ray structure of RIG-I RD the 5’-triphosphate interaction site could be mapped to a previously identified positively charged groove. Structural modeling of the homologous RD of MDA5 gave a rational for its lower affinity to RNA. Based on a comparison of enzymatic activities of several RIG-I truncation variants, a model for the transition from the inactive to the active state was postulated. In contrast to RIG-I, the molecular patterns which lead to MDA5-dependent anti-viral signaling are still insufficiently understood. Here it is shown that the dsRNA-mimic poly I:C is a potent activator of MDA5 ATPase activity in vitro. The ATPase activity is inhibited by V-protein, which stably binds to the N-terminal RecA domain of the MDA5 helicase domain. In summary, the results presented here broaden our understanding of virus sensing and pinpoint several intriguing avenues for future research. Protein conformation is critically linked to function and often controlled by interactions with regulatory factors. Here the selection of camelid-derived single-domain antibodies (nanobodies) that modulate the conformation and spectral properties of the green fluorescent protein (GFP)are reported. One nanobody could reversibly reduce GFP fluorescence about fivefold, whereas its displacement by a second nanobody caused a ten-fold increase. Structural analysis of GFP-nanobody complexes revealed that the two nanobodies induce subtle opposing changes in the chromophore environment leading to altered absorption properties. Unlike conventional antibodies, the small and stable nanobodies are functional in living cells. Nanobody induced changes were detected by ratio imaging and used to monitor protein expression and subcellular localization, and translocation events like the Tamoxifen-induced nuclear localization of estrogen receptor. This work demonstrates that protein conformations can be manipulated and studied with nanobodies in living cells.