The research performed in this thesis seeks primarily to elucidate the mechanism of B12 photoreceptors by using an integrated structural-dynamics and kinetic approach. The main system studied in this work is the CarH photoreceptor. CarH is a gene regulator photoreceptor that binds to operator DNA in bacteria in a tetrameric complex to inhibit gene expression. The suppressed gene controls carotenoid production that protects bacteria from sunlight damage.
The tetramer is stable in dark conditions and dissociates when it is exposed to light. The monomerization leads to a dissociation from the DNA, therefore allowing gene transcription to occur. The CarH photoreceptor hosts a B12 derivative chromophore (adenosylcobalamin, AdoCbl) which senses light, thus regulating gene expression. We focused on the chromophore binding domain (TtCBD) of the Thermus thermophilus CarH. We employed X-ray crystallography, specifically time-resolved serial femtosecond crystallography (TR-SFX) as the main technique, for studying structural changes in TtCBD in real time. The thesis addresses key challenges and applies methodologies across the entire experimental workflow for TR-SFX experiments, from sample preparation to data collection and processing. We report on the development of standardized protocols for obtaining seeded TtCBD microcrystals. We investigated and optimized different media (cellulose, lipidic cubic phase, grease) and methods for injecting and delivering microcrystals (high viscosity extruders, gas dynamic virtual nozzles, and fixed targets) while preserving their quality and photosensitivity. In terms of data collection, the thesis involves conducting static experiments at the ESRF synchrotron using macrocrystals of TtCBD and performing time-resolved experiments at various XFEL facilities (SACLA, LCLS, EuXFEL and SwissFEL). To further understand CarH photoactivation we have employed different techniques in collaboration, such as time-resolved solution scattering, transient absorption spectroscopy, and QM/MM calculations. We have elucidated structural and chemical changes following photon absorption up to tetramer dissociation on the ps to s timescale.
In the last part of this thesis, we introduce and characterize a novel viscous medium for high-viscosity extrusion (HVE) injection, based on guanosine derivatives hydrogels. Guanosine and guanosine-5’-monophosphate self-assemble into G-quartets, forming three-dimensional G-quadruplexes (G4) that create stable, viscous hydrogels with up to 99% water content. Our research focused on utilizing G4 hydrogels as a crystal-carrier matrix for SFX experiments. We investigated the hydrogel’s viscosity, jet generation, stability with protein crystallants, and ability to preserve microcrystals and enable photoactivation. We compared the G4 hydrogel’s X-ray background scattering at the ESRF ID29 beamline with other known media. Finally, we tested diffraction using this carrier medium with lysozyme microcrystals at SACLA, proving that we can obtain meaningful crystallographic data revealing the hydrogel’s effectiveness, highlighting its potential for TR-SFX studies.
This thesis has been carried out within a collaborative consortium involving laboratories from IBS Grenoble, the ESRF, the University of Manchester, and the University of Louisville.
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