%0 Generic
%A Nass Kovacs, Gabriela
%D 2019
%F heidok:26364
%R 10.11588/heidok.00026364
%T Light-energy conversion in rhodopsins studied by time-resolved serial femtosecond crystallography
%U https://archiv.ub.uni-heidelberg.de/volltextserver/26364/
%X Light is an important environmental factor used by light-sensitive proteins, photoreceptors, as a source of information or energy. In several classes of photoreceptors, photon absorption triggers the isomerization around a double bond in the light-sensitive chromophore. In rhodopsins, the isomerization of the chromophore retinal is one of the fastest light-triggered processes and it initiates different, protein-specific functions, spanning from vision and sleep regulation to light-energy conversion and phototaxis. The best characterized rhodopsin is bacteriorhodopsin (bR), a light-activated proton pump. Spectroscopic techniques were used to characterize both the ultrafast processes related to the isomerization, but also the later steps when the translocation of protons occurs. Structural knowledge of the later intermediates was provided mostly by X ray crystal structures of cryo-trapped bR or its mutants, contributing to a good understanding of the proton pumping steps. However, the ultrafast processes evolving on a sub-ps and ps time-scale are not amenable for time-resolved X-ray crystallography using synchrotron radiation, thus no structures of the ultrafast bR intermediates could be obtained. This changed with the advent of novel X-ray sources, the  X-ray free-electron lasers, which make time-resolved serial femtosecond crystallography (TR-SFX) on the sub-ps time-scale possible. This enables to address the most puzzling questions about the isomerization not only spectroscopically and computationally, but also structurally. Why is the isomerization of retinal in rhodopsins highly bond-specific and efficient, whereas it is neither specific nor efficient when retinal is free in solution? Is the protein affecting the isomerization reaction not only sterically, but also actively?  This work used TR-SFX to obtain structures of the ultrafast intermediates in bR in order to observe the structural changes in the retinal and in the protein on the sub-ps and ps time-scale. Large quantities of well-diffracting bR microcrystals were prepared in very viscous lipidic cubic phase. Time-resolved SFX experiments were performed only with liquid samples before the start of this thesis, therefore methods used for delivery of viscous samples in SFX needed to be adapted specifically for this time-resolved experiment.   The crystal structures obtained in the TR-SFX experiment on bR visualize the torsion of the isomerizing double bond in retinal. They also show oscillatory motion in the retinal and in specific protein residues and their distances to other residues or to ordered, functionally relevant water molecules. Changes in the distances in the internal hydrogen-bonded network of water molecules and protein residues are also observed. Similar to many TR-SFX experiments, this experiment was performed at very high pump laser excitation intensity, which can induce multiphoton processes, complicating the functional interpretation of the observations made. Unlike other TR-SFX experiments, this work acknowledges and addresses this caveat. Since the TR-SFX experiment could not be repeated at lower laser excitation intensity, additional spectroscopic and computational studies were carried out instead to gain more insight into multiphoton processes. These indeed provide new findings about decay channels in the multiphoton regime. Yet, it still remains open what the implications for the observations made in the TR-SFX structures are. In spite of that, a comparison of the TR-SFX observations with published spectroscopic and computational work performed in the single-photon regime shows remarkable similarities.   The insights obtained in this work pave the way for future TR-SFX experiments using optimal excitation conditions, which will clarify the relation of the observed structural changes to single-photon processes. This will allow judging whether the concerted motions observed in the retinal, residues and water molecules are part of the mechanism by which the protein actively controls the isomerization reaction of the chromophore. Furthermore, the methodological advances established here with the model system bR can now be directly applied to study other more challenging rhodopsins, such as the new family of anion-conducting channelrhodopsins (ACR). This work established insect-cell expression of an ACR protein and identified crystallization conditions yielding showers of microcrystals, which is the first step towards a future TR-SFX experiment.