Enzymes dramatically accelerate reaction rates, yet the detailed mechanisms of enzymatic catalysis, particularly the role of rapid electrostatic fluctuations, remain elusive. This study investigates pico- and nanosecond dynamics within dihydrofolate reductase (DHFR) from E. coli, focusing on substrate effects on electric field fluctuations during catalysis. Using molecular dynamics (MD) simulations, we examined structural and electrostatic changes in ligand-bound (DHFR•NADP•FOL) and unbound (apoform) states. Results show that ligand binding increases structural flexibility and dynamic behavior, indicated by higher radius of gyration (Rg), accelerates the decay of the autocorrelation function at sub-picosecond timescales, while slowing it at longer timescales, and increases root mean square deviation (RMSD) in specific regions. We find substantially enhanced localized electric field fluctuations around certain residues in the ligand-bound form but not in the apoform indicating that ligand binding significantly alters the electrostatic environment during the catalysis and potentially on energy barrier crossing events. Our findings highlight the importance of structural and electrostatic dynamics in enzyme function. Insights from this study can be applied in the design of effective enzyme inhibitors and engineered enzymes, advancing synthetic biology and protein engineering development.

X-ray crystallography has tremendously served structural biology by routinely providing high-resolution 3D structures of macromolecules. The extent of information encoded in the X-ray crystallography is proportional to which resolution the crystals diffract and the structure can be refined to. Therefore, there is a continuous effort to obtain high-quality crystals, especially for those proteins, which are considered difficult to crystallize into high-quality protein crystals of suitable sizes for X-ray crystallography. Efforts in enhancing the resolution in X-ray crystallography have also been made by optimizing crystallization protocols using external stimuli such as an electric field and magnetic field during the crystallization. Here, we present the feasibility of on-the-fly post-crystallization resolution enhancement of the protein crystal diffraction by applying a high-voltage electric field. The electric field between 2 and 11 kV/cm, which was applied after mounting the crystals in the beamline, resulted in the enhancement of the resolution. The crystal diffraction quality improved progressively with the exposure time. Moreover, we also find that upto defined electric field threshold, the protein structure remains largely unperturbed, a conclusion further supported by molecular dynamics simulations.

Organic semiconductors (OSCs) have emerged as promising materials for next-generation optoelectronic devices owing to their cost-effective processing and mechanical flexibility. Nevertheless, a thorough comprehension of ultrafast phenomena responsible for charge generation, transport, and recombination is imperative to fully exploit their potential. In this paper, we employ time-resolved spectroscopic ellipsometry (TRSE), an ultrafast, surface-sensitive, and non-invasive method, to explore these processes in thin films of a thiophene-linked diketopyrrolopyrrole (TDPP) derivative. While TRSE has proven effective for inorganic semiconductors, its application to organic materials remains unexplored. This technique is a crucial solution for measuring opaque samples or layers deposited on non-transparent substrates. TDPP molecule was selected for the measurement due to extensive research on this class of organic dyes using alternative spectroscopic techniques. After synthesis of TDPP via a multistep Suzuki-Miyaura cross coupling reaction, its thin film was deposited via vacuum sublimation. We demonstrate here the TRSE’s capability to elucidate optical phenomena in this material on a picosecond timescale, quantify the impacts of radiation damage, and derive kinetics without discernible sample damage. To support our findings, we measured kinetics using transient absorption spectroscopy (TAS) and validated the results. These findings highlight TRSE’s potential in understanding behaviors in organic materials and advancing their photophysics paving the way for better knowledge-based design and optimization of OSC-based devices.

Gallium sulphide (GaS) is an emerging monochalcogenide material that has recently attracted interest in optical technologies due to its tunable bandgap in the near-UV region. In this work, we employ in-situ, in-operando X-ray diffraction to investigate local atomic modifications in GaS induced by 400- nm femtosecond laser pulses. We identify the energy threshold at which irreversible structural changes occur and observe a laser-induced elongation of the unit cell along the c-axis. This elongation is expected to enhance the anisotropy of the material physical properties. Ab initio calculations further reveal that the experimentally observed ≈ 10% elongation along the c-axis leads to a transition from a direct to an indirect bandgap, accompanied by a bandgap increase of approximately 0.45 eV. Additional ab-initio optical simulations show that this structural transformation results in a nearly constant in-plane refractive index contrast of ∆n ≈ 0.1 across a wide spectral range, from the visible to the near-infrared, with negligible optical losses, which could be of interest for reconfigurable photonics applications.

Flavocytochrome c sulfide dehydrogenase (FCC) is an important enzyme of sulfur metabolism in sulfur-oxidizing bacteria, and its catalytic properties have been extensively studied. However, the ultrafast dynamics of FCC is not well understood. We present ultrafast transient absorption and fluorescence spectroscopy measurements to unravel the early events upon excitation of the heme and flavin chromophores embedded in the flavocytochrome c (FccAB) from the bacterium Thiocapsa roseopersicina. The fluorescence kinetics of FccAB suggests that the majority of the photoexcited species decay nonradiatively within the first few picoseconds. Transient absorption spectroscopy supports these findings by suggesting two major dynamic processes in FccAB, internal conversion occurring in about 400 fs and the vibrational cooling occurring in about 4 ps, mostly affecting the heme moiety.

Dynamic changes in protein glycosylation impact human health and disease progression. However, current resources that capture disease and phenotype information focus primarily on the macromolecules within the central dogma of molecular biology (DNA, RNA, proteins). To gain a better understanding of organisms, there is a need to capture the functional impact of glycans and glycosylation on biological processes. A workshop titled “Functional impact of glycans and their curation” was held in conjunction with the 16th Annual International Biocuration Conference to discuss ongoing worldwide activities related to glycan function curation. This workshop brought together subject matter experts, tool developers, and biocurators from over 20 projects and bioinformatics resources. Participants discussed four key topics for each of their resources: (i) how they curate glycan function-related data from publications and other sources, (ii) what type of data they would like to acquire, (iii) what data they currently have, and (iv) what standards they use. Their answers contributed input that provided a comprehensive overview of state-of-the-art glycan function curation and annotations. This report summarizes the outcome of discussions, including potential solutions and areas where curators, data wranglers, and text mining experts can collaborate to address current gaps in glycan and glycosylation annotations, leveraging each other’s work to improve their respective resources and encourage impactful data sharing among resources. Database URL: https://wiki.glygen.org/Glycan_Function_Workshop_2023

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We report the first application of broadband time-resolved pump-probe ellipsometry to study the ultrafast dynamics of the photoinduced insulator-to-metal transition (IMT) in vanadium dioxide (VO2) thin films driven by 35 fs laser pulses. This novel technique enables the direct measurement of the time-resolved evolution of the complex pseudodielectric function of VO(2 )during the IMT. We have identified distinct thermal and nonthermal dynamics in the photoinduced IMT, which critically depends on the pump wavelength and fluence, while providing a detailed temporal and spectral phase map. A comparison of the pseudodielectric function of the VO2 thin film during thermally and photoinduced phase transitions reveals that the primary differences in the IMT pathways occur within the first picosecond after the pump, driven by nonequilibrium dynamics in this ultrafast time scale. The ultrafast spectroscopic ellipsometry introduced in this work offers a complementary probe to study phase changes in condensed matter and emerging photonic device materials.

With the emergence of ultrafast X-ray sources, interest in following fast processes in small molecules and macromolecules has increased. Most of the current research into ultrafast structural dynamics of macromolecules uses X-ray free-electron lasers. In parallel, small-scale laboratory-based laser-driven ultrafast X-ray sources are emerging. Continuous development of these sources is underway, and as a result many exciting applications are being reported. However, because of their low flux, such sources are not commonly used to study the structural dynamics of macromolecules. This article examines the feasibility of time-resolved powder diffraction of macromolecular microcrystals using a laboratory-scale laser-driven ultrafast X-ray source.

X-ray crystallography is an established tool to probe the structure of macromolecules with atomic resolution. Compared with alternative techniques such as single-particle cryo-electron microscopy and micro-electron diffraction, X-ray crystallography is uniquely suited to room-temperature studies and for obtaining a detailed picture of macromolecules subjected to an external electric field (EEF). The impact of an EEF on proteins has been extensively explored through single-crystal X-ray crystallography, which works well with larger high-quality protein crystals. This article introduces a novel design for a 3D-printed in situ crystallization plate that serves a dual purpose: fostering crystal growth and allowing the concurrent examination of the effects of an EEF on crystals of varying sizes. The plate's compatibility with established X-ray crystallography techniques is evaluated.

Acoustic phonons are generated by the arrival of a femtosecond laser pulse to the surface of a material. By femtosecond pump-probe ellipsometry on thin films and bulk materials, we could discern between different types of transient phenomena including the ones due to the propagation of these photo-induced acoustic waves.

This paper discusses the fundamentals, applications, potential and limitations of polarized light reflection techniques for the characterization of phase-change materials (PCMs). These techniques include spectroscopic ellipsometry, time-resolved ellipsometry and imaging ellipsometry as well as polarimetry. We explore the ca-pabilities of spectroscopic ellipsometry in the determination of the extinction coefficient of PCMs and the ca-pabilities of imaging ellipsometry to characterize PCMs. We show that ellipsometry is capable of more than the determination of thickness and optical properties, and it can be exploited to gain information about crystalli-zation/amorphization kinetics and mapping anisotropies.

We report the current status of laser-plasma accelerator-based secondary X-ray sources at ELI beamlines.

The realization of compact X-ray sources is one of the most intriguing applications of laser-plasma based electron acceleration. These sources based on the oscillation of short micron-sized bunches of relativistic electrons provide femtosecond X-ray pulses that are collimated, bright, and partially coherent. The state-of-the-art laser plasma X-ray sources can provide photon flux of over 1011 photons/shot. The photon flux can further be enhanced with the availability of high repetition rate, high-power lasers, providing capacities complementary to the large scale facilities such as synchrotrons and X-ray free-electron lasers. Even though the optimization of such sources has been underway for the last two decades, their applications in material and biological sciences are still emerging, which entail the necessity of a user-oriented X-ray beamlines. Based on this concept, a high-power-laser-based user-oriented X-ray source is being developed at ELI Beamlines. This article reports on the ELI Gammatron beamline and presents an overview of the research accessible with the ultrashort hard X-ray pulses at the ELI Gammatron beamline.

X-ray diffraction microscopy (XDM) is an established lens-less imaging method extensively practiced at synchrotrons and X-ray free-electron lasers (XFELs). XDM is broadly operated in two different modes: scanning and non-scanning. The non-scanning mode of operation in XDM is commonly called coherent diffraction imaging (CDI) and has been the key research direction of many XFEL facilities. This method typically images objects smaller than the size of the illumination, which precludes the imaging of a large group of samples physically larger than the illumination. Furthermore, satisfying this requirement at X-ray free-electron lasers tremendously reduces the volume of practically useful data, leading the experimental scheme to be less efficient. Such a limitation can be circumvented by using a uniform illumination probe rather than the traditional Gaussian-focused probe from the X-ray focusing optics. Here in this article, we report a numerical study on the design of an optical element to generate uniform X-ray illumination and its application to the CDI. We demonstrate the benefits of such illumination in imaging objects that are larger than the illumination size and in improving the efficiency of the experimental scheme overall.

A new generation of small-scale ultrafast X-ray sources is rapidly emerging. Laser-driven betatron radiation represents an important class of such ultrafast X-ray sources. With the sources driving towards maturity, many important applications in material and biological sciences are expected to be carried out. While the last decade mainly focused on the optimization of the source properties, the development of such sources into user-oriented beamlines in order to explore the potential applications has recently taken off and is expected to grow rapidly. An important aspect in the realization of such beamlines will be the implementation of proper X-ray optics. Here, we present the design of a multi-lane X-ray mirror as a versatile focusing device covering a wide spectral range of betatron X-rays. The expected photon flux in the focal plane of such optics was also estimated through geometrical simulations.

We report on the status of a users' end-station, MAC: a Multipurpose station for Atomic, molecular and optical sciences and Coherent diffractive imaging, designed for studies of structure and dynamics of matter in the femtosecond time-domain. MAC is located in the E1 experimental hall on the high harmonic generation (HHG) beamline of the ELI Beamlines facility. The extreme ultraviolet beam from the HHG beamline can be used at the MAC end-station together with a synchronized pump beam (which will cover the NIR/Vis/UV or THz range) for time-resolved experiments on different samples. Sample delivery systems at the MAC end-station include a molecular beam, a source for pure or doped clusters, ultrathin cylindrical or flat liquid jets, and focused beams of substrate-free nanoparticles produced by an electrospray or a gas dynamic virtual nozzle combined with an aerodynamic lens stack. We further present the available detectors: electron/ion time-of-flight and velocity map imaging spectrometers and an X-ray camera, and discuss future upgrades: a magnetic bottle electron spectrometer, production of doped nanodroplets and the planned developments of beam capabilities at the MAC end-station.

Green compositions and processes for fabrication of dual- and multiloaded nanocarriers with an antioxidant functionality and neuro-protective, cardioprotective, antiviral, and antiproliferative activities are of broad interest for innovations in pharmaceutics and nutraceutics. Coencapsulation of curcumin (studied as a key multipurpose phytochemical antioxidant) with omega-3 polyunsaturated fatty acids (PUFAs) (studied as active ingredients of natural fish oil) may increase the oxidative stability of self-assembled formulations aiming at the development of "food drugs" and prevention of disease progression in various pathological states. The objective of this work is to prepare self-assembled lyotropic liquid crystalline nanostructures as dual-loaded biodegradable carriers of omega-3 PUFA-fish oil and curcumin. A detailed structural phase diagram of a ternary [monoolein-PEGylated lipid mixture]/[fish oil-curcumin]/water system is created. A composition-mediated switch between nanostructures of different topologies and polymorphic states is achieved through varying the ratios between the amphiphilic monoglyceride ingredient, fish oil, and water, which yielded cubic, sponge, and lamellar mesophases of tunable nanoscale repeat spacings. Synchrotron small-angle X-ray scattering (SAXS) studies are performed with the lyotropic liquid crystalline nanostructures along compositional dilution lines. The temperature effect is examined at 22 and 5 degrees C with regard to preparation conditions and mesophase stability on storage. Bulk mesophases are dispersed into lipid nanopartides at 22 degrees C, the structures and topologies of which are revealed by SAXS and cryo-transmission electron microscopy imaging. New knowledge about the controlled multicomponent supramolecular assembly and the achieved stabilization of low-temperature cubic phases (hydrated in 5 wt % D-(+)-glucose) should facilitate the development of cost-effective, stable, and safe delivery systems of weakly soluble natural antioxidant compounds coencapsulated with omega-3 PUFA oils.

Time-resolved in-house macromolecular crystallography is primarily limited by the capabilities of the in-house X-ray sources. These sources can only provide a time-averaged structure of the macromolecules. A significant effort has been made in the development of in-house laser-driven ultrafast X-ray sources, with one of the goals as realizing the visualization of the structural dynamics of macromolecules at a very short timescale within the laboratory-scale infrastructure. Most of such in-house ultrafast X-ray sources are operated at high repetition rates and usually deliver very low flux. Therefore, the necessity of a detector that can operate at the repetition rate of the laser and perform extremely well under low flux conditions is essential. Here, we present experimental results demonstrating the usability of the hybrid-pixel detectors, such as Eiger X 1M, and provide experimental proof that they can be successfully operated to collect macromolecular crystallographic data up to a detector frame rate of 3 kHz from synchrotron sources. Our results also show that the data reduction and structural analysis are successful at such high frame rates and fluxes as low as 10(8) photons/s, which is comparable to the values expected from a typical laser-driven X-ray source.