Time-Resolved Serial-Femtosecond Crystallography of the G Protein-Coupled Receptor Rhodopsin

Start Date
06-03-2020 13:30
End Date
06-03-2020 14:30
Room 500 - 501, Central Building
Speaker's name
Thomas GRUHL
Speaker's institute
Institut Paul Scherrer, Villigen, Suisse
Contact name
Claudine Roméro
Host name
Gordon Leonard
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Cells can interact with the environment through proteins embedded in their membrane. Due to their diversity and prevalence, G protein-coupled receptors (GPCRs) are one of the most important groups of such membrane receptors. Within this large protein family, light-sensitive rhodopsins are responsible for photoreception and vision. As part of its mechanism of light detection, rhodopsin triggers one of the fastest chemical reactions in biology, the cis-trans photoisomerization of a double bond in the chromophore retinal, a cofactor derived from vitamin A covalently bound to the protein. After photoactivation, rhodopsin undergoes conformational changes leading to an activated "META" state able to bind the next partner in the signalling cascade, the G protein transducin. Rhodopsin and the structurally related bacteriorhodopsin are considered prototypes for the study of the molecular mechanisms of photoactivation in membrane proteins. However, while rhodopsins are eukaryotic GPCRs, bacteriorhodopsin is a bacterial proton pump, and different mechanisms are therefore expected.

Recently, X-ray free electron lasers (XFEL) have revolutionized the field of structural biology by enabling the dynamic study of protein structural changes in a timescale from fs to ms. In pump-probe serial femtosecond crystallography XFEL experiments, the X-ray pulse for acquiring structural data follows a ‘trigger’ (e.g. photoactivation) after specified time delays, allowing to measure conformational changes in the protein along its activation at very high temporal and spatial resolution. This method requires a constant renewal of protein crystals, as, after diffracting, they are immediately destroyed by the brilliant X-ray pulses.

Using this technique, the photoactivation mechanism of the proton pump bacteriorhodopsin has been recently determined by obtaining X-ray diffraction data in a time scale from femtoseconds to milliseconds at the LCLS (Linac Coherent Light Source) and SACLA (SPring-8 Angstrom Compact Free Electron Laser) FELs. This work has revealed subtle but decisive events of charge transfer and conformational changes leading ultimately to the pumping of protons. On the other hand, for eukaryotic rhodopsins, only ‘static’ structures of dark and active states have been characterised by X-ray crystallography in cryogenic conditions. The aim of this thesis is to use time-resolved X-ray crystallography in FELs to study rhodopsin dynamics. Here, I present protocols for its purification, crystallisation, data processing, and analysis.

First, we optimised existing purification protocols and established the first crystallisation of wild-type mammalian rhodopsin in lipidic cubic phase (the environment of choice for most serial crystallography experiments of membrane proteins). Crystals obtained by this method allowed us to solve the structure of rhodopsin to a resolution of 1.8 Å. They were also suitable for time-resolved studies, and we collected data of eight photoactivated intermediates ranging from fs to ms. This thesis focuses on the analysis of two time-points, 1 and 100 picoseconds, crucial in two early events of retinal photoactivation: retinal isomerisation and thermal relaxation of the protein. The 1 ps data was collected in a pilot experiment at SwissFEL as one of the first in house user beamtime. Our data shows a fully isomerised retinal already at 1 ps, in which the rotation of a methyl group forces a water to move, resulting in structural rearrangements of nearby residues (E181, Y268 and Y191/192). In summary, we show for the first time activation events in rhodopsin at the edge of ultrafast time scales, opening the possibility for studies at the fs regime. With the analysis of the remaining data points, further insights of the activation mechanism of GPCRs will be gained, which will lead to a better understanding of this important family of membrane proteins.

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