Time-resolved X-ray Liquidography Research Articles

Structural dynamics of protein-protein association involved in the light-induced transition of Avena sativa LOV2 protein

The Light-oxygen-voltage-sensing domain (LOV) superfamily, found in enzymes and signal transduction proteins, plays a crucial role in converting light signals into structural signals, mediating various biological mechanisms. While time-resolved spectroscopic studies have revealed the dynamics of the LOV-domain chromophore’s electronic structures, understanding the structural changes in the protein moiety, particularly regarding light-induced dimerization, remains challenging. Here, we utilize time-resolved X-ray liquidography to capture the light-induced dimerization of Avena sativa LOV2. Our analysis unveils that dimerization occurs within milliseconds after the unfolding of the A’α and Jα helices in the microsecond time range. Notably, our findings suggest that protein-protein interactions (PPIs) among the β-scaffolds, mediated by helix unfolding, play a key role in dimerization. In this work, we offer structural insights into the dimerization of LOV2 proteins following structural changes in the A’α and Jα helices, as well as mechanistic insights into the protein-protein association process driven by PPIs.

Serial X-ray liquidography: multi-dimensional assay framework for exploring biomolecular structural dynamics with microgram quantities

Understanding protein structure and kinetics under physiological conditions is crucial for elucidating complex biological processes. While time-resolved (TR) techniques have advanced to track molecular actions, their practical application in biological reactions is often confined to reversible photoreactions within limited experimental parameters due to inefficient sample utilization and inflexibility of experimental setups. Here, we introduce serial X-ray liquidography (SXL), a technique that combines time-resolved X-ray liquidography with a fixed target of serially arranged microchambers. SXL breaks through the previously mentioned barriers, enabling microgram-scale TR studies of both irreversible and reversible reactions of even a non-photoactive protein. We demonstrate its versatility in studying a wide range of biological reactions, highlighting its potential as a flexible and multi-dimensional assay framework for kinetic and structural characterization. Leveraging X-ray free-electron lasers and micro-focused X-ray pulses promises further enhancements in both temporal resolution and minimizing sample quantity. SXL offers unprecedented insights into the structural and kinetic landscapes of molecular actions, paving the way for a deeper understanding of complex biological processes.

The carbon-iodine bond cleavage and isomerization of iodoform visualized with femtosecond X-ray liquidography.

Iodoform (CHI3) has garnered significant attention for its unique ability to induce photo-cyclopropanation of olefins by releasing an iodine radical through C-I bond cleavage. However, the detailed mechanism underlying CHI3 photodissociation is still not fully understood. Here, we elucidate the ultrafast structural dynamics of CHI3 upon photoexcitation using femtosecond time-resolved X-ray liquidography (fs-TRXL) at an X-ray free-electron laser facility. The fs-TRXL data was decomposed into the isotropic and anisotropic data. The isotropic data reveal that the formation of CHI2 and I radicals upon photolysis precedes the emergence of iso-CHI2-I. After a short induction period, two competing geminate recombination pathways of CHI2 and I radicals take place: one pathway leads to the recovery of CHI3, while the other results in the formation of iso-CHI2-I. Additionally, the anisotropic data show how the transient anisotropic distribution of both the species formed upon photoexcitation and the ground-state species depleted upon photoexcitation decays through rotational dephasing. Furthermore, the observed structural dynamics of CHI3 has distinctive differences with that of BiI3, which can be attributed to differences in their central moieties, CH and Bi. Our findings provide insights into the photoinduced reaction dynamics of CHI3, enhancing the understanding of its role in photochemical reactions.

Cerium Photocatalyst in Action: Structural Dynamics in the Presence of Substrate Visualized via Time-Resolved X-ray Liquidography.

[Ce(III)Cl6]3-, with its earth-abundant metal element, is a promising photocatalyst facilitating carbon-halogen bond activation. Still, the structure of the reaction intermediate has yet to be explored. Here, we applied time-resolved X-ray liquidography (TRXL), which allows for direct observation of the structural details of reaction intermediates, to investigate the photocatalytic reaction of [Ce(III)Cl6]3-. Structural analysis of the TRXL data revealed that the excited state of [Ce(III)Cl6]3- has Ce-Cl bonds that are shorter than those of the ground state and that the Ce-Cl bond further contracts upon oxidation. In addition, this study represents the first application of TRXL to both photocatalyst-only and photocatalyst-and-substrate samples, providing insights into the substrate's influence on the photocatalyst's reaction dynamics. This study demonstrates the capability of TRXL in elucidating the reaction dynamics of photocatalysts under various conditions and highlights the importance of experimental determination of the structures of reaction intermediates to advance our understanding of photocatalytic mechanisms.

Projection to extract the perpendicular component (PEPC) method for extracting kinetics from time-resolved data

Time-resolved x-ray liquidography (TRXL) is a potent method for investigating the structural dynamics of chemical and biological reactions in the liquid phase. It has enabled the extraction of detailed structural aspects of various dynamic processes, the molecular structures of intermediates, and kinetics of reactions across a wide range of systems, from small molecules to proteins and nanoparticles. Proper data analysis is key to extracting the information of the kinetics and structural dynamics of the studied system encrypted in the TRXL data. In typical TRXL data, the signals from solute scattering, solvent scattering, and solute–solvent cross scattering are mixed in the q-space, and the solute kinetics and solvent hydrodynamics are mixed in the time domain, thus complicating the data analysis. Various methods developed so far generally require prior knowledge of the molecular structures of candidate species involved in the reaction. Because such information is often unavailable, a typical data analysis often involves tedious trial and error. To remedy this situation, we have developed a method named projection to extract the perpendicular component (PEPC), capable of removing the contribution of solvent kinetics from TRXL data. The resulting data then contain only the solute kinetics, and, thus, the solute kinetics can be easily determined. Once the solute kinetics is determined, the subsequent data analysis to extract the structural information can be performed with drastically improved convenience. The application of the PEPC method is demonstrated with TRXL data from the photochemistry of two molecular systems: [Au(CN)2−]3 in water and CHI3 in cyclohexane.

Extracting Kinetics and Thermodynamics of Molecules without Heavy Atoms via Time-Resolved Solvent Scattering Signals.

Time-resolved X-ray liquidography (TRXL) has emerged as a powerful technique for studying the structural dynamics of small molecules and macromolecules in liquid solutions. However, TRXL has limited sensitivity for small molecules containing light atoms only, whose signal has lower contrast compared with the signal from solvent molecules. Here, we present an alternative approach to bypass this limitation by detecting the change in solvent temperature resulting from a photoinduced reaction. Specifically, we analyzed the heat dynamics of TRXL data obtained from p-hydroxyphenacyl diethyl phosphate (HPDP). This analysis enabled us to experimentally determine the number of intermediates and their respective enthalpy changes, which can be compared to theoretical enthalpies to identify the intermediates. This work demonstrates that TRXL can be used to uncover the kinetics and reaction pathways for small molecules without heavy atoms even if the scattering signal from the solute molecules is buried under the strong solvent scattering signal.

Publisher's Note: “Structural dynamics of proteins explored via time-resolved x-ray liquidography” [Chem. Phys. Rev. 3, 041304 (2022)

First Page

Structural dynamics of proteins explored via time-resolved x-ray liquidography

The structure of a protein is closely related to its biological function. In this regard, structural changes, as well as static structures, have been scrutinized as essential elements in understanding and controlling the function of a protein. In particular, the structural change in the solution phase needs to be elucidated to properly understand protein functions under physiological conditions. Time-resolved x-ray liquidography (TRXL), also known as time-resolved x-ray solution scattering, has attracted attention as a powerful experimental method for studying the structural dynamics of proteins in the solution phase. Initially, TRXL was used to study the structural dynamics of small molecules in the solution phase, and later, its application was extended to probe the structural changes in proteins. Via TRXL, structural changes ranging from large quaternary movements to subtle rearrangements of the tertiary structures have been successfully elucidated. In this review, we introduce various studies using TRXL to investigate the structural dynamics of proteins. These include early TRXL studies on model systems, those on photoreceptor proteins, and recent studies using stimuli beyond the direct photoexcitation of proteins.

Photoactivation of triosmium dodecacarbonyl at 400 nm probed with time-resolved X-ray liquidography.

The photoactivation mechanism of Os3(CO)12 at 400 nm is examined with time-resolved X-ray liquidography. The data reveal two pathways: the vibrational relaxation following an internal conversion to the electronic ground state and the ligand dissociation to form Os3(CO)11 with a ligand vacancy at the axial position.

Structural Dynamics of C2F4I2 in Cyclohexane Studied via Time-Resolved X-ray Liquidography.

The halogen elimination of 1,2-diiodoethane (C2H4I2) and 1,2-diiodotetrafluoroethane (C2F4I2) serves as a model reaction for investigating the influence of fluorination on reaction dynamics and solute–solvent interactions in solution-phase reactions. While the kinetics and reaction pathways of the halogen elimination reaction of C2H4I2 were reported to vary substantially depending on the solvent, the solvent effects on the photodissociation of C2F4I2 remain to be explored, as its reaction dynamics have only been studied in methanol. Here, to investigate the solvent dependence, we conducted a time-resolved X-ray liquidography (TRXL) experiment on C2F4I2 in cyclohexane. The data revealed that (ⅰ) the solvent dependence of the photoreaction of C2F4I2 is not as strong as that observed for C2H4I2, and (ⅱ) the nongeminate recombination leading to the formation of I2 is slower in cyclohexane than in methanol. We also show that the molecular structures of the relevant species determined from the structural analysis of TRXL data provide an excellent benchmark for DFT calculations, especially for investigating the relevance of exchange-correlation functionals used for the structural optimization of haloalkanes. This study demonstrates that TRXL is a powerful technique to study solvent dependence in the solution phase.

Optical Kerr Effect of Liquid Acetonitrile Probed by Femtosecond Time-Resolved X-ray Liquidography.

Optical Kerr effect (OKE) spectroscopy is a method that measures the time-dependent change of the birefringence induced by an optical laser pulse using another optical laser pulse and has been used often to study the ultrafast dynamics of molecular liquids. Here we demonstrate an alternative method, femtosecond time-resolved X-ray liquidography (fs-TRXL), where the microscopic structural motions related to the OKE response can be monitored using a different type of probe, i.e., X-ray solution scattering. By applying fs-TRXL to acetonitrile and a dye solution in acetonitrile, we demonstrate that different types of molecular motions around photoaligned molecules can be resolved selectively, even without any theoretical modeling, based on the anisotropy of two-dimensional scattering patterns and extra structural information contained in the q-space scattering data. Specifically, the dynamics of reorientational (libration and orientational diffusion) and translational (interaction-induced motion) motions are captured separately by anisotropic and isotropic scattering signals, respectively. Furthermore, the two different types of reorientational motions are distinguished from each other by their own characteristic scattering patterns and time scales. The measured time-resolved scattering signals are in excellent agreement with the simulated scattering signals based on a molecular dynamics simulation for plausible molecular configurations, providing the detailed structural description of the OKE response in liquid acetonitrile.

Filming ultrafast roaming-mediated isomerization of bismuth triiodide in solution

Roaming reaction, defined as a reaction yielding products via reorientational motion in the long-range region (3 – 8 Å) of the potential, is a relatively recently proposed reaction pathway and is now regarded as a universal mechanism that can explain the unimolecular dissociation and isomerization of various molecules. The structural movements of the partially dissociated fragments originating from the frustrated bond fission at the onset of roaming, however, have been explored mostly via theoretical simulations and rarely observed experimentally. Here, we report an investigation of the structural dynamics during a roaming-mediated isomerization reaction of bismuth triiodide (BiI3) in acetonitrile solution using femtosecond time-resolved x-ray liquidography. Structural analysis of the data visualizes the atomic movements during the roaming-mediated isomerization process including the opening of the Bi-Ib-Ic angle and the closing of Ia-Bi-Ib-Ic dihedral angle, each by ~40°, as well as the shortening of the Ib···Ic distance, following the frustrated bond fission.

Revealing structural dynamics of proteins with time-resolved X-ray liquidography

Revealing structural dynamics of proteins with time-resolved X-ray liquidography

Femtosecond X-ray Liquidography Visualizes Wavepacket Trajectories in Multidimensional Nuclear Coordinates for a Bimolecular Reaction.

ConspectusVibrational wavepacket motions on potential energy surfaces are one of the critical factors that determine the reaction dynamics of photoinduced reactions. The motions of vibrational wavepackets are often discussed in the interpretation of observables measured with various time-resolved vibrational or electronic spectroscopies but mostly in terms of the frequencies of wavepacket motions, which are approximated by normal modes, rather than the actual positions of the wavepacket. Although the time-dependent positions (that is, the trajectory) of wavepackets are hypothesized or drawn in imagined or calculated potential energy surfaces, it is not trivial to experimentally determine the trajectory of wavepackets, especially in multidimensional nuclear coordinates for a polyatomic molecule. Recently, we performed a femtosecond X-ray liquidography (solution scattering) experiment on a gold trimer complex (GTC), [Au(CN)2-]3, in water at X-ray free-electron lasers (XFELs) and elucidated the time-dependent positions of vibrational wavepackets from the Franck-Condon region to equilibrium structures on both excited and ground states in the course of the formation of covalent bonds between gold atoms.Bond making is an essential process in chemical reactions, but it is challenging to keep track of detailed atomic movements associated with bond making because of its bimolecular nature that requires slow diffusion of two reaction parties to meet each other. Bond formation in the solution phase has been elusive because the diffusion of the reactants limits the reaction rate of a bimolecular process, making it difficult to initiate and track the bond-making processes with an ultrafast time resolution. In principle, if the bimolecular encounter can be controlled to overcome the limitation caused by diffusion, the bond-making processes can be tracked in a time-resolved manner, providing valuable insight into the bimolecular reaction mechanism. In this regard, GTC offers a good model system for studying the dynamics of bond formation in solution. Au(I) atoms in GTC exhibit a noncovalent aurophilic interaction, making GTC an aggregate complex without any covalent bond. Upon photoexcitation of GTC, an electron is excited from an antibonding orbital to a bonding orbital, leading to the formation of covalent bonds among Au atoms. Since Au atoms in the ground state of GTC are located in close proximity within the same solvent cage, the formation of Au-Au covalent bonds occurs without its reaction rate being limited by diffusion through the solvent.Femtosecond time-resolved X-ray liquidography (fs-TRXL) data revealed that the ground state has an asymmetric bent structure. From the wavepacket trajectory determined in three-dimensional nuclear coordinates (two internuclear distances and one bond angle), we found that two covalent bonds are formed between three Au atoms of GTC asynchronously. Specifically, one covalent bond is formed first for the shorter Au-Au pair (of the asymmetric and bent ground-state structure) in 35 fs, and subsequently, the other covalent bond is formed for the longer Au-Au pair within 360 fs. The resultant trimer complex has a symmetric and linear geometry, implying the occurrence of bent-to-linear transformation concomitant with the formation of two equivalent covalent bonds, and exhibits vibrations that can be unambiguously assigned to specific normal modes based on the wavepacket trajectory, even without the vibrational frequencies provided by quantum calculation.

Gold Nanoparticle Formation via X-ray Radiolysis Investigated with Time-Resolved X-ray Liquidography

We report the generation of gold nanoparticles (AuNPs) from the aqueous solution of chloro(2,2′,2″-terpyridine)gold(III) ion ([Au(tpy)Cl]2+) through X-ray radiolysis and optical excitation at a synchrotron. The original purpose of the experiment was to investigate the photoinduced structural changes of [Au(tpy)Cl]2+ upon 400 nm excitation using time-resolved X-ray liquidography (TRXL). Initially, the TRXL data did not show any signal that would suggest structural changes of the solute molecule, but after an induction time, the TRXL data started to show sharp peaks and valleys. In the early phase, AuNPs with two types of morphology, dendrites, and spheres, were formed by the reducing action of hydrated electrons generated by the X-ray radiolysis of water, thereby allowing the detection of TRXL data due to the laser-induced lattice expansion and relaxation of AuNPs. Along with the lattice expansion, the dendritic and spherical AuNPs were transformed into smaller, raspberry-shaped AuNPs of a relatively uniform size via ablation by the optical femtosecond laser pulse used for the TRXL experiment. Density functional theory calculations confirm that the reduction potential of the metal complex relative to the hydration potential of X-ray-generated electrons determines the facile AuNP formation observed for [Au(tpy)Cl]2+.

Estimating signal and noise of time-resolved X-ray solution scattering data at synchrotrons and XFELs.

Elucidating the structural dynamics of small molecules and proteins in the liquid solution phase is essential to ensure a fundamental understanding of their reaction mechanisms. In this regard, time-resolved X-ray solution scattering (TRXSS), also known as time-resolved X-ray liquidography (TRXL), has been established as a powerful technique for obtaining the structural information of reaction intermediates and products in the liquid solution phase and is expected to be applied to a wider range of molecules in the future. A TRXL experiment is generally performed at the beamline of a synchrotron or an X-ray free-electron laser (XFEL) to provide intense and short X-ray pulses. Considering the limited opportunities to use these facilities, it is necessary to verify the plausibility of a target experiment prior to the actual experiment. For this purpose, a program has been developed, referred to as S-cube, which is short for a Solution Scattering Simulator. This code allows the routine estimation of the shape and signal-to-noise ratio (SNR) of TRXL data from known experimental parameters. Specifically, S-cube calculates the difference scattering curve and the associated quantum noise on the basis of the molecular structure of the target reactant and product, the target solvent, the energy of the pump laser pulse and the specifications of the beamline to be used. Employing a simplified form for the pair-distribution function required to calculate the solute-solvent cross term greatly increases the calculation speed as compared with a typical TRXL data analysis. Demonstrative applications of S-cube are presented, including the estimation of the expected TRXL data and SNR level for the future LCLS-II HE beamlines.

Fate of transient isomer of CH2I2: Mechanism and origin of ionic photoproducts formation unveiled by time-resolved x-ray liquidography.

Diiodomethane, CH2I2, in a polar solvent undergoes a unique photoinduced reaction whereby I2 - and I3 - are produced from its photodissociation, unlike for other iodine-containing haloalkanes. While previous studies proposed that homolysis, heterolysis, or solvolysis of iso-CH2I-I, which is a major intermediate of the photodissociation, can account for the formation of I2 - and I3 -, there has been no consensus on its mechanism and no clue for the reason why those negative ionic species are not observed in the photodissociation of other iodine-containing chemicals in the same polar solvent, for example, CHI3, C2H4I2, C2F4I2, I3 -, and I2. Here, using time-resolved X-ray liquidography, we revisit the photodissociation mechanism of CH2I2 in methanol and determine the structures of all transient species and photoproducts involved in its photodissociation and reveal that I2 - and I3 - are formed via heterolysis of iso-CH2I-I in the photodissociation of CH2I2 in methanol. In addition, we demonstrate that the high polarity of iso-CH2I-I is responsible for the unique photochemistry of CH2I2.

Structural Dynamics of Bismuth Triiodide in Solution Triggered by Photoinduced Ligand-to-Metal Charge Transfer.

Bismuth triiodide, BiI3, is one of the simplest bismuth halides, which have recently attracted considerable attention because of their promising properties. Here, we investigate the structural dynamics of a photoinduced reaction of BiI3 in solution phase using time-resolved X-ray liquidography (TRXL) and density functional theory (DFT) and time-dependent DFT (TDDFT) calculations. The photoreaction was initiated by excitation at 400 nm, which corresponds to the ligand-to-metal charge-transfer transition. The detailed structures and kinetic profiles of all relevant intermediate species from the TRXL data show that the trigonal planar structure of BiI3, which is predicted to be the most stable structure of the lowest excited state by TDDFT calculation, was not observed, and the photoreaction proceeds via two parallel pathways within the time resolution of 100 ps: (i) isomer formation to produce iso-BiI2-I, which relaxes back to the ground-state structure, and (ii) dissociation into BiI2· and I· radicals, which nongeminately recombine to generate ground-state BiI3 and I2.

Correction to "Direct Observation of a Transiently Formed Isomer During Iodoform Photolysis in Solution by Time-Resolved X-ray Liquidography".

Correction to "Direct Observation of a Transiently Formed Isomer During Iodoform Photolysis in Solution by Time-Resolved X-ray Liquidography".

Direct Observation of a Transiently Formed Isomer During Iodoform Photolysis in Solution by Time-Resolved X-ray Liquidography.

Photolysis of iodoform (CHI3) in solution has been extensively studied, but its reaction mechanism remains elusive. In particular, iso-iodoform (iso-CHI2-I) is formed as a product of the photolysis reaction, but its detailed structure is not known, and whether it is a major intermediate species has been controversial. Here, by using time-resolved X-ray liquidography, we determined the reaction mechanism of CHI3 photodissociation in cyclohexane as well as the structure of iso-CHI2-I. Both iso-CHI2-I and CHI2 radical were found to be formed within 100 ps with a branching ratio of 40:60. Iodine radicals (I), formed during the course of CHI3 photolysis, recombine nongeminately with either CHI2 or I. Based on our structural analysis, the I-I distance and the C-I-I angle of iso-CHI2-I were determined to be 2.922 ± 0.004 Å and 133.9 ± 0.8°, respectively.