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.

Oligomerization processes limit photoactivation and recovery of the orange carotenoid protein.

The orange carotenoid protein (OCP) is a photoactive protein involved in cyanobacterial photoprotection by quenching of the excess of light-harvested energy. The photoactivation mechanism remains elusive, in part due to absence of data pertaining to the timescales over which protein structural changes take place. It also remains unclear whether or not oligomerization of the dark-adapted and light-adapted OCP could play a role in the regulation of its energy-quenching activity. Here, we probed photoinduced structural changes in OCP by a combination of static and time-resolved X-ray scattering and steady-state and transient optical spectroscopy in the visible range. Our results suggest that oligomerization partakes in regulation of the OCP photocycle, with different oligomers slowing down the overall thermal recovery of the dark-adapted state of OCP. They furthermore reveal that upon non-photoproductive excitation a numbed state forms, which remains in a non-photoexcitable structural state for at least ≈0.5 µs after absorption of a first photon.

In situ structural kinetics of picosecond laser-induced heating and fragmentation of colloidal gold spheres.

Fragmentation of colloidal 54 nm gold nanoparticles by picosecond laser pulses is recorded by time-resolved X-ray scattering, giving access to structural dynamics down to a 80 ps resolution. Lattice temperature and energy dissipation have been quantified to verify that the maximum applied fluence of 1800 J m-2 heats up the particles close to boiling. Already within 30 ns, particles with significantly lower particle sizes of 2 to 3 nm are detected, which hints towards an ultrafast process either by a thermal phase explosion or Coulomb instability. An arrested growth is observed on a microsecond time scale resulting in a final particle size of 3-4 nm with high yield. In this context, the fragmentation in a NaCl/NaOH solution seems to limit growth by electrostatic stabilization of fragments, whereas it does not modify the initial product sizes. The laser-induced fragmentation process is identified as a single-step, instantaneous reaction.

Evidence for laser-induced homogeneous oriented ice nucleation revealed via pulsed x-ray diffraction.

The induction of homogeneous and oriented ice nucleation has to date not been achieved. Here, we report induced nucleation of ice from millimeter sized supercooled water drops illuminated by ns-optical laser pulses well below the ionization threshold making use of particular laser beam configurations and polarizations. Employing a 100 ps synchrotron x-ray pulse 100 ns after each laser pulse, an unambiguous correlation was observed between the directions and the symmetry of the laser fields and that of the H-bonding arrays of the induced ice crystals. Moreover, an analysis of the x-ray diffraction data indicates that, in the main, the induced nucleation of ice is homogeneous at temperatures well above the observed and predicted values for supercooled water.

Demonstration of a picosecond Bragg switch for hard X-rays in a synchrotron-based pump-probe experiment.

A benchmark experiment is reported that demonstrates the shortening of hard X-ray pulses in a synchrotron-based optical pump-X-ray probe measurement. The pulse-shortening device is a photoacoustic Bragg switch that reduces the temporal resolution of an incident X-ray pulse to approximately 7.5 ps. The Bragg switch is employed to monitor propagating sound waves in nanometer thin epitaxial films. From the experimental data, the pulse duration, diffraction efficiency and switching contrast of the device can be inferred. A detailed efficiency analysis shows that the switch can deliver up to 109 photons s-1 in high-repetition-rate synchrotron experiments.

Hysteresis in the temperature dependence of the IR bending vibration of deeply cooled confined water.

Using Fourier Transform Infrared (FTIR) spectroscopy, we investigate the temperature dependence of the bending vibrations of water confined in the pores of a silica hydrogel in the temperature interval of 270-180 K. We also investigate the presence of thermal hysteresis by cooling and reheating temperature scans. The results clearly show the presence, at about 230 K, of a crossover in the temperature dependence of the IR spectra; moreover, the presence of hysteresis is clearly demonstrated. By comparing FTIR data with neutron diffraction data and previous calorimetric data on the same samples, we conclude that the crossover and the hysteretical behavior do not involve a water glass transition or crystallization but are related to a first-order-like liquid-liquid transition.

Functional and dysfunctional conformers of human neuroserpin characterized by optical spectroscopies and Molecular Dynamics.

Neuroserpin (NS) is a serine protease inhibitor (SERPIN) involved in different neurological pathologies, including the Familial Encephalopathy with Neuroserpin Inclusion Bodies (FENIB), related to the aberrant polymerization of NS mutants. Here we present an in vitro and in silico characterization of native neuroserpin and its dysfunctional conformation isoforms: the proteolytically cleaved conformer, the inactive latent conformer, and the polymeric species. Based on circular dichroism and fluorescence spectroscopy, we present an experimental validation of the latent model and highlight the main structural features of the different conformers. In particular, emission spectra of aromatic residues yield distinct conformational fingerprints, that provide a novel and simple spectroscopic tool for selecting serpin conformers in vitro. Based on the structural relationship between cleaved and latent serpins, we propose a structural model for latent NS, for which an experimental crystallographic structure is lacking. Molecular Dynamics simulations suggest that NS conformational stability and flexibility arise from a spatial distribution of intramolecular salt-bridges and hydrogen bonds.

The Monod-Wyman-Changeux allosteric model accounts for the quaternary transition dynamics in wild type and a recombinant mutant human hemoglobin.

The acknowledged success of the Monod-Wyman-Changeux (MWC) allosteric model stems from its efficacy in accounting for the functional behavior of many complex proteins starting with hemoglobin (the paradigmatic case) and extending to channels and receptors. The kinetic aspects of the allosteric model, however, have been often neglected, with the exception of hemoglobin and a few other proteins where conformational relaxations can be triggered by a short and intense laser pulse, and monitored by time-resolved optical spectroscopy. Only recently the application of time-resolved wide-angle X-ray scattering (TR-WAXS), a direct structurally sensitive technique, unveiled the time scale of hemoglobin quaternary structural transition. In order to test the generality of the MWC kinetic model, we carried out a TR-WAXS investigation in parallel on adult human hemoglobin and on a recombinant protein (HbYQ) carrying two mutations at the active site [Leu(B10)Tyr and His(E7)Gln]. HbYQ seemed an ideal test because, although exhibiting allosteric properties, its kinetic and structural properties are different from adult human hemoglobin. The structural dynamics of HbYQ unveiled by TR-WAXS can be quantitatively accounted for by the MWC kinetic model. Interestingly, the main structural change associated with the R-T allosteric transition (i.e., the relative rotation and translation of the dimers) is approximately 10-fold slower in HbYQ, and the drop in the allosteric transition rate with ligand saturation is steeper. Our results extend the general validity of the MWC kinetic model and reveal peculiar thermodynamic properties of HbYQ. A possible structural interpretation of the characteristic kinetic behavior of HbYQ is also discussed.

Physical Regimes and Mechanisms of Picosecond Laser Fragmentation of Gold Nanoparticles in Water from X-ray Probing and Atomistic Simulations.

Laser fragmentation in liquids has emerged as a promising green chemistry technique for changing the size, shape, structure, and phase composition of colloidal nanoparticles, thus tuning their properties to the needs of practical applications. The advancement of this technique requires a solid understanding of the mechanisms of laser-nanoparticle interactions that lead to the fragmentation. While theoretical studies have made impressive practical and mechanistic predictions, their experimental validation is required. Hence, using the picosecond laser fragmentation of Au nanoparticles in water as a model system, the transient melting and fragmentation processes are investigated with a combination of time-resolved X-ray probing and atomistic simulations. The direct comparison of the diffraction profiles predicted in the simulations and measured in experiments has revealed a sequence of several nonequilibrium processes triggered by the laser irradiation. At low laser fluences, in the regime of nanoparticle melting and resolidification, the results provide evidence of a transient superheating of crystalline nanoparticles above the melting temperature. At fluences about three times the melting threshold, the fragmentation starts with evaporation of Au atoms and their condensation into small satellite nanoparticles. As fluence increases above five times the melting threshold, a transition to a rapid (explosive) phase decomposition of superheated nanoparticles into small liquid droplets and vapor phase atoms is observed. The transition to the phase explosion fragmentation regime is signified by prominent changes in the small-angle X-ray scattering profiles measured in experiments and calculated in simulations. The good match between the experimental and computational diffraction profiles gives credence to the physical picture of the cascade of thermal fragmentation regimes revealed in the simulations and demonstrates the high promise of the joint tightly integrated computational and experimental efforts.

Ultrafast and persistent photoinduced phase transition at room temperature monitored by streaming powder diffraction.

Ultrafast photoinduced phase transitions at room temperature, driven by a single laser shot and persisting long after stimuli, represent emerging routes for ultrafast control over materials' properties. Time-resolved studies provide fundamental mechanistic insight into far-from-equilibrium electronic and structural dynamics. Here we study the photoinduced phase transformation of the Rb0.94Mn0.94Co0.06[Fe(CN)6]0.98 material, designed to exhibit a 75 K wide thermal hysteresis around room temperature between MnIIIFeII tetragonal and MnIIFeIII cubic phases. We developed a specific powder sample streaming technique to monitor by ultrafast X-ray diffraction the structural and symmetry changes. We show that the photoinduced polarons expand the lattice, while the tetragonal-to-cubic photoinduced phase transition occurs within 100 ps above threshold fluence. These results are rationalized within the framework of the Landau theory of phase transition as an elastically-driven and cooperative process. We foresee broad applications of the streaming powder technique to study non-reversible and ultrafast dynamics.

Tracking the structural dynamics of proteins with time-resolved X-ray solution scattering.

Relevant events during protein function such as ligand binding/release and interaction with substrates or with light are often accompanied by out-of-equilibrium structural dynamics. Time-resolved experimental techniques have been developed to follow protein structural changes as they happen in real time after a given reaction-triggering event. Time-resolved X-ray solution scattering is a promising approach that bears structural sensitivity with temporal resolution in the femto-to-millisecond time range, depending on the X-ray source characteristics and the triggering method. Here we present the basic principles of the technique together with a description of the most relevant results recently published and a discussion on the computational methods currently developed to achieve a structural interpretation of the time-resolved X-ray solution scattering experimental data.

X-ray beam quality after a mirror reflection: Experimental and simulated results for a toroidal mirror in a 4 th generation storage-ring beamline.

Background: The surface errors found in X-ray mirrors constitute a limiting factor for preserving beam quality. This is particularly important when the X-ray beam has low emittance and a significant coherence fraction, like in newly upgraded synchrotron storage rings. Methods: We studied the fringes observed in the image of an undulator-produced X-ray beam reflected by a high-quality toroidal mirror. The measurements and simulations were performed using different conditions: a photon beam either monochromatic or with large bandwidth, reflected by a mirror with variable curvature. Results: The experimental data are compared with up-to-date simulation including partial coherence. Conclusions: The observed fringes in the unfocused beam correlate with low spatial frequency structures in mirror profiles, irrespective of beam coherence. Both classical ray tracing and partially coherent simulations through coherent mode decomposition are confirmed as accurate methods for such simulations.

In this study, researchers focused on the surface errors found in X-ray mirrors and their impact on beam quality. These errors can be problematic, especially when dealing with X-ray beams coming from low emittance (a measure of beam size and divergence) electron beam sources and a significant coherence fraction (indicating the level of wavefront coherence). The researchers specifically investigated the fringes observed in the image of an X-ray beam produced by an undulator and reflected by a high-quality toroidal mirror. They conducted measurements and simulations under different conditions, such as using a monochromatic photon beam or one with a wide range of wavelengths, and varying the curvature of the mirror.

On the molecular structure of human neuroserpin polymers.

The polymerization of serpins is at the root of a large class of diseases; the molecular structure of serpin polymers has been recently debated. In this work, we study the polymerization kinetics of human neuroserpin by Fourier Transform Infra Red spectroscopy and by time-lapse Size Exclusion Chromatography. First, we show that two distinct neuroserpin polymers, formed at 45 and 85°C, display the same isosbestic points in the Amide I' band, and therefore share common secondary structure features. We also find a concentration independent polymerization rate at 45°C suggesting that the polymerization rate-limiting step is the formation of an activated monomeric species. The polymer structures are consistent with a model that predicts the bare insertion of portions of the reactive center loop into the A ß-sheet of neighboring serpin molecule, although with different extents at 45 and 85°C.

Modeling difference x-ray scattering observations from an integral membrane protein within a detergent micelle.

Time-resolved x-ray solution scattering (TR-XSS) is a sub-field of structural biology, which observes secondary structural changes in proteins as they evolve along their functional pathways. While the number of distinct conformational states and their rise and decay can be extracted directly from TR-XSS experimental data recorded from light-sensitive systems, structural modeling is more challenging. This step often builds from complementary structural information, including secondary structural changes extracted from crystallographic studies or molecular dynamics simulations. When working with integral membrane proteins, another challenge arises because x-ray scattering from the protein and the surrounding detergent micelle interfere and these effects should be considered during structural modeling. Here, we utilize molecular dynamics simulations to explicitly incorporate the x-ray scattering cross term between a membrane protein and its surrounding detergent micelle when modeling TR-XSS data from photoactivated samples of detergent solubilized bacteriorhodopsin. This analysis provides theoretical foundations in support of our earlier approach to structural modeling that did not explicitly incorporate this cross term and improves agreement between experimental data and theoretical predictions at lower x-ray scattering angles.

Tracking the structural dynamics of proteins in solution using time-resolved wide-angle X-ray scattering.

We demonstrate tracking of protein structural changes with time-resolved wide-angle X-ray scattering (TR-WAXS) with nanosecond time resolution. We investigated the tertiary and quaternary conformational changes of human hemoglobin under nearly physiological conditions triggered by laser-induced ligand photolysis. We also report data on optically induced tertiary relaxations of myoglobin and refolding of cytochrome c to illustrate the wide applicability of the technique. By providing insights into the structural dynamics of proteins functioning in their natural environment, TR-WAXS complements and extends results obtained with time-resolved optical spectroscopy and X-ray crystallography.

Tracking the ATP-binding response in adenylate kinase in real time.

The biological function of proteins is critically dependent on dynamics inherent to the native structure. Such structural dynamics obey a predefined order and temporal timing to execute the specific reaction. Determination of the cooperativity of key structural rearrangements requires monitoring protein reactions in real time. In this work, we used time-resolved x-ray solution scattering (TR-XSS) to visualize structural changes in the Escherichia coli adenylate kinase (AdK) enzyme upon laser-induced activation of a protected ATP substrate. A 4.3-ms transient intermediate showed partial closing of both the ATP- and AMP-binding domains, which indicates a cooperative closing mechanism. The ATP-binding domain also showed local unfolding and breaking of an Arg131-Asp146 salt bridge. Nuclear magnetic resonance spectroscopy data identified similar unfolding in an Arg131Ala AdK mutant, which refolded in a closed, substrate-binding conformation. The observed structural dynamics agree with a "cracking mechanism" proposed to underlie global structural transformation, such as allostery, in proteins.

Charge transfer driven by ultrafast spin transition in a CoFe Prussian blue analogue.

Photoinduced charge-transfer is an important process in nature and technology and is responsible for the emergence of exotic functionalities, such as magnetic order for cyanide-bridged bimetallic coordination networks. Despite its broad interest and intensive developments in chemistry and material sciences, the atomic-scale description of the initial photoinduced process, which couples intermetallic charge-transfer and spin transition, has been debated for decades; it has been beyond reach due to its extreme speed. Here we study this process in a prototype cyanide-bridged CoFe system by femtosecond X-ray and optical absorption spectroscopies, enabling the disentanglement of ultrafast electronic and structural dynamics. Our results demonstrate that it is the spin transition that occurs first on the Co site within ~50 fs, and it is this that drives the subsequent Fe-to-Co charge-transfer within ~200 fs. This study represents a step towards understanding and controlling charge-transfer-based functions using light.

Two latent and two hyperstable polymeric forms of human neuroserpin.

Human neuroserpin (hNS) is a serine protease inhibitor that belongs to the serpin superfamily and is expressed in nervous tissues. The serpin fold is generally characterized by a long exposed loop, termed the reactive center loop, that acts as bait for the target protease. Intramolecular insertion of the reactive center loop into the main serpin ß-sheet leads to the serpin latent form. As with other known serpins, hNS pathological mutants have been shown to accumulate as polymers composed of quasi-native protein molecules. Although hNS polymerization has been intensely studied, a general agreement about serpin polymer organization is still lacking. Here we report a biophysical characterization of native hNS that is shown to undergo two distinct conformational transitions, at 55°C and 85°C, both leading to distinct latent and polymeric species. The latent and polymer hNS forms obtained at 45°C and 85°C differ in their chemical and thermal stabilities; furthermore, the hNS polymers also differ in size and morphology. Finally, the 85°C polymer shows a higher content of intermolecular ß-sheet interactions than the 45°C polymer. Together, these results suggest a more complex conformational scenario than was previously envisioned, and, in a general context, may help reconcile the current contrasting views on serpin polymerization.

Tracking Ca2+ ATPase intermediates in real time by x-ray solution scattering.

Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) transporters regulate calcium signaling by active calcium ion reuptake to internal stores. Structural transitions associated with transport have been characterized by x-ray crystallography, but critical intermediates involved in the accessibility switch across the membrane are missing. We combined time-resolved x-ray solution scattering (TR-XSS) experiments and molecular dynamics (MD) simulations for real-time tracking of concerted SERCA reaction cycle dynamics in the native membrane. The equilibrium [Ca2]E1 state before laser activation differed in the domain arrangement compared with crystal structures, and following laser-induced release of caged ATP, a 1.5-ms intermediate was formed that showed closure of the cytoplasmic domains typical of E1 states with bound Ca2+ and ATP. A subsequent 13-ms transient state showed a previously unresolved actuator (A) domain arrangement that exposed the ADP-binding site after phosphorylation. Hence, the obtained TR-XSS models determine the relative timing of so-far elusive domain rearrangements in a native environment.

Taking a snapshot of the triplet excited state of an OLED organometallic luminophore using X-rays.

OLED technology beyond small or expensive devices requires light-emitters, luminophores, based on earth-abundant elements. Understanding and experimental verification of charge transfer in luminophores are needed for this development. An organometallic multicore Cu complex comprising Cu-C and Cu-P bonds represents an underexplored type of luminophore. To investigate the charge transfer and structural rearrangements in this material, we apply complementary pump-probe X-ray techniques: absorption, emission, and scattering including pump-probe measurements at the X-ray free-electron laser SwissFEL. We find that the excitation leads to charge movement from C- and P- coordinated Cu sites and from the phosphorus atoms to phenyl rings; the Cu core slightly rearranges with 0.05 Å increase of the shortest Cu-Cu distance. The use of a Cu cluster bonded to the ligands through C and P atoms is an efficient way to keep structural rigidity of luminophores. Obtained data can be used to verify computational methods for the development of luminophores.