Light-energy conversion in rhodopsins studied by time-resolved serial femtosecond crystallography

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

Three-dimensional view of ultrafast dynamics in photoexcited bacteriorhodopsin

Bacteriorhodopsin (bR) is a light-driven proton pump. The primary photochemical event upon light absorption is isomerization of the retinal chromophore. Here we used time-resolved crystallography at an X-ray free-electron laser to follow the structural changes in multiphoton-excited bR from 250 femtoseconds to 10 picoseconds. Quantum chemistry and ultrafast spectroscopy were used to identify a sequential two-photon absorption process, leading to excitation of a tryptophan residue flanking the retinal chromophore, as a first manifestation of multiphoton effects. We resolve distinct stages in the structural dynamics of the all-trans retinal in photoexcited bR to a highly twisted 13-cis conformation. Other active site sub-picosecond rearrangements include correlated vibrational motions of the electronically excited retinal chromophore, the surrounding amino acids and water molecules as well as their hydrogen bonding network. These results show that this extended photo-active network forms an electronically and vibrationally coupled system in bR, and most likely in all retinal proteins

Influence of pump laser fluence on ultrafast structural changes in myoglobin

High-intensity femtosecond pulses from an X-ray free-electron laser enable pump probe experiments for investigating electronic and nuclear changes during light-induced reactions. On time scales ranging from femtoseconds to milliseconds and for a variety of biological systems, time-resolved serial femtosecond crystallography (TR-SFX) has provided detailed structural data for light-induced isomerization, breakage or formation of chemical bonds and electron transfer 1 . However, all ultra-fast TR-SFX studies to date have employed such high pump laser energies that several photons were nominally absorbed per chromophore 2-14 . As multiphoton absorption may force the protein response into nonphysiological pathways, it is of great concern 15 whether this experimental approach 16 allows valid inferences to be drawn vis-à-vis biologically relevant single-photon-induced reactions 17 . Here we describe ultrafast pump-probe SFX experiments on photodissociation of carboxymyoglobin, showing that different pump laser fluences yield markedly different results. In particular, the dynamics of structural changes and observed indicators of the mechanistically important coherent oscillations of the Fe-CO bond distance (predicted by recent quantum wavepacket dynamics 15 ) are seen to depend strongly on pump laser energy. Our results confirm both the feasibility and necessity of performing TR-SFX pump probe experiments in the linear photoexcitation regime. We consider this to be a starting point for reassessing design and interpretation of ultrafast TR-SFX pump probe experiments 16 such that biologically relevant insight emerges

Influence of pump laser fluence on ultrafast myoglobin structural dynamics

International audienceHigh-intensity femtosecond pulses from an X-ray free-electron laser enable pump–probe experiments for the investigation of electronic and nuclear changes during light-induced reactions. On timescales ranging from femtoseconds to milliseconds and for a variety of biological systems, time-resolved serial femtosecond crystallography (TR-SFX) has provided detailed structural data for light-induced isomerization, breakage or formation of chemical bonds and electron transfer 1,2 . However, all ultrafast TR-SFX studies to date have employed such high pump laser energies that nominally several photons were absorbed per chromophore 3–17 . As multiphoton absorption may force the protein response into non-physiological pathways, it is of great concern 18,19 whether this experimental approach 20 allows valid conclusions to be drawn vis-à-vis biologically relevant single-photon-induced reactions 18,19 . Here we describe ultrafast pump–probe SFX experiments on the photodissociation of carboxymyoglobin, showing that different pump laser fluences yield markedly different results. In particular, the dynamics of structural changes and observed indicators of the mechanistically important coherent oscillations of the Fe–CO bond distance (predicted by recent quantum wavepacket dynamics 21 ) are seen to depend strongly on pump laser energy, in line with quantum chemical analysis. Our results confirm both the feasibility and necessity of performing ultrafast TR-SFX pump–probe experiments in the linear photoexcitation regime. We consider this to be a starting point for reassessing both the design and the interpretation of ultrafast TR-SFX pump–probe experiments 20 such that mechanistically relevant insight emerges

Photoswitching mechanism of a fluorescent protein revealed by time-resolved crystallography and transient absorption spectroscopy.

International audienceReversibly switchable fluorescent proteins (RSFPs) serve as markers in advanced fluorescence imaging. Photoswitching from a non-fluorescent off-state to a fluorescent on-state involves trans-to-cis chromophore isomerization and proton transfer. Whereas excited-state events on the ps timescale have been structurally characterized, conformational changes on slower timescales remain elusive. Here we describe the off-to-on photoswitching mechanism in the RSFP rsEGFP2 by using a combination of time-resolved serial crystallography at an X-ray free-electron laser and ns-resolved pump-probe UV-visible spectroscopy. Ten ns after photoexcitation, the crystal structure features a chromophore that isomerized from trans to cis but the surrounding pocket features conformational differences compared to the final on-state. Spectroscopy identifies the chromophore in this ground-state photo-intermediate as being protonated. Deprotonation then occurs on the μs timescale and correlates with a conformational change of the conserved neighbouring histidine. Together with a previous excited-state study, our data allow establishing a detailed mechanism of off-to-on photoswitching in rsEGFP2

Three-dimensional view of ultrafast dynamics in photoexcited bacteriorhodopsin

International audienceBacteriorhodopsin (bR) is a light-driven proton pump. The primary photochemical event uponlight absorption is isomerization of the retinal chromophore. Here we used time-resolvedcrystallography at an X-ray free-electron laser to follow the structural changes inmultiphoton-excited bR from 250 femtoseconds to 10 picoseconds. Quantum chemistry andultrafast spectroscopy were used to identify a sequential two-photon absorption process,leading to excitation of a tryptophan residueflanking the retinal chromophore, as afirstmanifestation of multiphoton effects. We resolve distinct stages in the structural dynamics ofthe all-transretinal in photoexcited bR to a highly twisted 13-cisconformation. Other activesite sub-picosecond rearrangements include correlated vibrational motions of the electro-nically excited retinal chromophore, the surrounding amino acids and water molecules as wellas their hydrogen bonding network. These results show that this extended photo-activenetwork forms an electronically and vibrationally coupled system in bR, and most likely in allretinal proteins

Rational Control of Off‐State Heterogeneity in a Photoswitchable Fluorescent Protein Provides Switching Contrast Enhancement**

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MHz data collection of a microcrystalline mixture of different jack bean proteins

Design Type(s)protocol testing objectiveMeasurement Type(s)protein structure dataTechnology Type(s)x ray crystallographyFactor Type(s)Sample Characteristic(s)Canavalia Machine-accessible metadata file describing the reported data (ISA-Tab format

Megahertz data collection from protein microcrystals at an X-ray free-electron laser

The European X-ray free-electron laser (EuXFEL) in Hamburg is the first megahertz (MHz) repetition rate XFEL. Here the authors use lysozyme crystals and microcrystals from jack bean proteins and demonstrate that damage-free high quality data can be collected at a MHz repetition rate

De novo determination of mosquitocidal Cry11Aa and Cry11Ba structures from naturally-occurring nanocrystals

Cry11Aa and Cry11Ba are the two most potent toxins produced by mosquitocidal Bacillus thuringiensis subsp. israelensis and jegathesan , respectively. The toxins naturally crystallize within the host; however, the crystals are too small for structure determination at synchrotron sources. Therefore, we applied serial femtosecond crystallography at X-ray free electron lasers to in vivo -grown nanocrystals of these toxins. The structure of Cry11Aa was determined de novo using the single-wavelength anomalous dispersion method, which in turn enabled the determination of the Cry11Ba structure by molecular replacement. The two structures reveal a new pattern for in vivo crystallization of Cry toxins, whereby each of their three domains packs with a symmetrically identical domain, and a cleavable crystal packing motif is located within the protoxin rather than at the termini. The diversity of in vivo crystallization patterns suggests explanations for their varied levels of toxicity and rational approaches to improve these toxins for mosquito control