Two-dimensional confinement for generating thin single crystals for applications in time-resolved electron diffraction and spectroscopy: an intramolecular proton transfer study

Thin single organic crystals (≤1 μm) with large area (≥100 × 100 μm2) are desirable to explore photoinduced processes using ultrafast spectroscopy and electron-diffraction. Here, we present a general method based on spatial confinement to grow such crystals using the prototypical proton transfer system, 1,5-dihydroxyanthraquinone, as an example, and provide the protocol for optically characterizing structural dynamics to enable proper assignments using diffraction methods

Peptide Mass Spectra from Micrometer-Thick Ice Films Produced with Femtosecond Pulses

We present a cryogenic mass spectrometry protocol with the capability to detect peptides in the attomole dilution range from ice films. Our approach employs femtosecond laser pulses and implements neither substrate modification nor proton donor agents in the aqueous solution, known to facilitate analyte detection in mass spectrometry. In a systematic study, we investigated the impact of temperature, substrate composition, and irradiation wavelength (513 and 1026 nm) on the bradykinin signal onset. Our findings show that substrate choice and irradiation wavelength have a minor impact on signal intensity once the preparation protocol is optimized. However, if the temperature is increased from −140 to 0 °C, which is accompanied by ice film thinning, a somehow complex picture of analyte desorption and ionization is recognizable, which has not been described in the literature yet. Under cryogenic conditions (−140 °C), obtaining a signal is only possible from isolated sweet spots across the film. If the thin ice film is between −100 and −70 °C of temperature, these sweet spots appear more frequently. Ice sublimation triggered by temperatures above −70 °C leads to an intense and robust signal onset that could be maintained for several hours. In addition to the above findings, we notice that a vibrant fragmentation pattern produced is strikingly similar with both wavelengths. Our findings suggest that while following an optimized protocol, femtosecond mass spectrometry has excellent potential to analyze small organic molecules and peptides with a mass range of up to 2.5 kDa in aqueous solution without any matrix, as employed in matrix-assisted laser desorption/ionization (MALDI) or any substrate surface modification, found in surface-assisted laser desorption/ionization (SALDI)

An electropneumatic cleaning device for piezo-actuator-driven picolitre-droplet dispensers

Recently, we introduced the liquid application method for time-resolved analyses (LAMA). The time-consuming cleaning cycles required for the substrate solution exchange and storage of the sensitive droplet-dispenser nozzles present practical challenges. In this work, a dispenser cleaning system for the semi-automated cleaning of the piezo-actuator-driven picolitre-droplet dispensers required for LAMA is introduced to streamline typical workflows

SPITROBOT-2 advances time-resolved cryo-trapping crystallography to under 25 ms

We previously introduced the SPITROBOT, a protein crystal plunging system that enables reaction quenching via cryo-trapping with a time resolution in the millisecond range. Here we present the next generation, SPITROBOT-2, as an integrated benchtop device, condensed to approximately an A4 footprint. The user experience has been enhanced by the integration of a guiding beam and a sample-switch dial, to optimise sample exchange operations. This is complemented by a light-indicated liquid nitrogen level sensor, ensuring enhanced reliability. Moreover, a fully automated shutter shields the liquid nitrogen from the humidified environment, improving sample integrity. These improvements reduce the net sample preparation time to approximately three minutes per sample. Most importantly, the cryo-trapping delay time has been reduced to 23 milliseconds, making SPITROBOT-2 twice as fast as the previous generation. This further expands the number of target systems that can be addressed by cryo-trapping time-resolved crystallography. We demonstrate successful cryo-trapping via ligand binding and conformational changes using 12 crystal structures of three independent model systems: xylose isomerase, human insulin and bacteriophage T4 lysozyme. Taken together, these improvements increase the convenient access to cryo-trapping, time-resolved X-ray crystallography empowering the MX community with efficient tools to advance research in structural biology

Probing the modulation of enzyme kinetics by multi-temperature, time-resolved serial crystallography

The vast majority of protein structures are determined at cryogenic temperatures, which are far from physiological conditions. Nevertheless, it is well established that temperature is an essential thermodynamic parameter for understanding the conformational dynamics and functionality of proteins in their native environments. Time-resolved crystallography is a technique that aims to elucidate protein function by examining structural alterations during processes such as ligand binding, catalysis, or allostery. However, this approach is typically conducted under ambient conditions, which may obscure crucial conformational states, that are only visible at physiological temperatures. In this study, we directly address the interplay between protein structure and activity via a method that enables multi-temperature, time-resolved serial crystallography experiments in a temperature window from below 10 °C to above 70 °C. Via this 5D-SSX, time-resolved experiments can now be carried out at physiological temperatures and with long time delays, providing insights into protein function and enzyme catalysis. Our findings demonstrate the temperature-dependent modulation of turnover kinetics for the mesophilic β-lactamase CTX-M-14 and the thermophilic enzyme xylose isomerase, within the full protein structure

Intermolecular Interactions in Crystals Modulate Intramolecular Excited State Proton Transfer Reactions

Proton transfer is a fundamental process underlying chemical and biological phenomena, and its dynamics are significantly influenced by the surrounding environment. This paper studies the excited state intramolecular proton transfer (ESIPT) process, which is crucial to the photostability of hydroxyanthraquinone-based pigments through efficient energy dissipation, by investigating how crystalline packing influences photoinduced proton transfer dynamics in single crystals of dihydroxyanthraquinone (DHAQ) constitutional isomers. Comparing the proton transfer dynamics in crystalline and solution phases, we show substantial differences due to the crystalline environment, particularly in the 1,4- and 1,5-DHAQ isomers. These isomers show intermolecular hydrogen bonding within the crystal lattice, resulting in larger excitonic couplings, which significantly alters their reaction pathways compared to their behavior in solution. We show that 1,8-DHAQ, which does not form intermolecular hydrogen bonds in the crystal, shows minimal changes in dynamics between the phases. In contrast, in the case of 1,4-DHAQ, intermolecular interactions within the molecular crystal phase open up an ESIPT relaxation channel, which is not observed in the solution phase. These findings highlight the critical role of crystal packing in modulating proton transfer dynamics and offer insights into how molecular packing can be strategically manipulated to control and optimize reaction pathways in solid-state environments

The HARE chip for efficient time-resolved serial synchrotron crystallography

Serial synchrotron crystallography (SSX) is an emerging technique for static and time-resolved protein structure determination. Using specifically patterned silicon chips for sample delivery, the `hit-and-return' (HARE) protocol allows for efficient time-resolved data collection. The specific pattern of the crystal wells in the HARE chip provides direct access to many discrete time points. HARE chips allow for optical excitation as well as on-chip mixing for reaction initiation, making a large number of protein systems amenable to time-resolved studies. Loading of protein microcrystals onto the HARE chip is streamlined by a novel vacuum loading platform that allows fine-tuning of suction strength while maintaining a humid environment to prevent crystal dehydration. To enable the widespread use of time-resolved serial synchrotron crystallography (TR-SSX), detailed technical descriptions of a set of accessories that facilitate TR-SSX workflows are provided

Extreme timescale core-level spectroscopy with tailored XUV pulses

A new approach for few-femtosecond time-resolved photoelectron spectroscopy in condensed matter that balances the combined needs for both temporal and energy resolution is demonstrated. Here, the method is designed to investigate a prototypical Mott insulator, tantalum disulphide (1T-TaS2), which transforms from its charge-density-wave ordered Mott insulating state to a conducting state in a matter of femtoseconds. The signature to be observed through the phase transition is a charge-density-wave induced splitting of the Ta 4f core-levels, which can be resolved with sub-eV spectral resolution. Combining this spectral resolution with few-femtosecond time resolution enables the collapse of the charge ordered Mott state to be clocked. Precise knowledge of the sub-20-femtosecond dynamics will provide new insight into the physical mechanism behind the collapse and may reveal Mott physics on the timescale of electronic hopping.Comment: 20 pages, 6 figure