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Towards structural studies of Hepadnavirus subviral particles using wheat germ cell-free expression and solid-state NMR

Abstract : Structural studies of eukaryotic membrane proteins are of prime importance but notoriously difficult as they not only necessitate an efficient and practical overexpression system that allows for membrane protein expression in a biologically relevant folding, but also a structural technique that you can easily combine with the chosen protein production system. In vitro cell-free systems, due to their modulable nature, are particularly suited for membrane protein expression. Furthermore, they now established themselves as a viable alternative to conventional cell-based expression, notably because of considerable advances in robustness and efficiency. Amongst them, the wheat germ cell-free production system (WG-CFPS) proved to be the most efficient for production of eukaryotic membrane proteins, and allows for efficient and specific isotope labeling. This makes it particularly convenient for Nuclear Magnetic Resonance (NMR), and more specifically solid-state NMR which is particularly appropriate for membrane protein studies and macromolecular assemblies. Thanks to very recent advances that lead to a drastic reduction of the quantity of protein needed, solid-state NMR is now compatible with WG-CFPS, creating a powerful tool for structural studies of macromolecular assemblies and membrane proteins. In this work, these two techniques are combined for the production and study of the envelope proteins from the duck Hepatitis B Virus (DHBV), that belongs to the Hepadnaviridae family. These viruses are able to secrete active virions, but also particles composed only of envelope proteins, which are called subviral particles (SVPs). In the first part, we show here that the DHBV small envelope protein (DHBs S) is produced as soluble in mg amounts using WG-CFPS. Even more, the protein forms SVPs upon translation, and is thus expressed in a biologically relevant form. After SVPs disassembly, the protein displays a mostly -helical folding, which is characteristic of a well-folded protein, and also very similar to the secondary structure of an assembly-incompetent mutant. After further isolation by ultracentrifugation on a sucrose gradient, the SVPs were sedimented in a 0.7 mm rotor and observed by solid-state NMR. Very promising hNH 2D spectra, with a good signal, were obtained. They display numerous isolated peaks and a resolution alike to other sedimented membrane proteins observed by solid-state NMR. Moreover, superimposition of the DHBs S spectrum with simulated spectra from proteins with extreme secondary structure content confirms that the protein is mostly -helical in the context of the SVPs. Nonetheless, the signal still needs to be improved in order to perform the experiments necessary for in-depth structural analysis. To that end, sample optimization assays were conducted. On the one hand, protein yield improvement, by the use of a commercial wheat germ extract, and SVPs stabilization, by incubation with KSCN, were tried. On the other hand, different methods for SVPs purification were tested, including PEG6000 or ammonium sulfate precipitation, incubation at high temperature, contaminant removal with an ultrafiltration device, affinity or size-exclusion purification as well as tests of particles disassembly, purification followed by SVPs reconstitution in lipids. Finally, amino-acid specific isotopic labeling of DHBs S was evaluated. In the second part, we could show extended possibilities of WG-CFPS through expression of DHBV large envelope protein (DHBs L). In vivo, the protein undergo specific phosphorylation as well as alternative translation, and we could show that it is also the case upon wheat germ cell-free expression. We also tested coexpression of DHBs S, DHBs L and of the DHBV capsid in order to assess the possibility of DHBs L inclusion in SVPs, or even complete virion reconstitution, which could even augment WG-CFPS possibilities. Ultimately, we also detail some critical parameters for SVPs formation in the WG-CFPS
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Guillaume David. Towards structural studies of Hepadnavirus subviral particles using wheat germ cell-free expression and solid-state NMR. Biochemistry, Molecular Biology. Université de Lyon, 2019. English. ⟨NNT : 2019LYSE1336⟩. ⟨tel-02613779v2⟩

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