Search

The development of serial crystallography over the last decade at XFELs and synchrotrons has produced a renaissance in room-temperature macromolecular crystallography (RT-MX), and fostered many technical and methodological breakthroughs designed to study phenomena occurring in proteins on the picosecond-to-second timescale. However, there are components of protein dynamics that occur in much slower regimes, of which the study could readily benefit from state-of-the-art RT-MX. Here, the room-temperature structural study of the relaxation of a reaction intermediate at a synchrotron, exploiting a handful of single crystals, is described. The intermediate in question is formed in microseconds during the photoreaction of the LOV2 domain of phototropin 2 from Arabidopsis thaliana , which then decays in minutes. This work monitored its relaxation in the dark using a fast-readout EIGER X 4M detector to record several complete oscillation X-ray diffraction datasets, each of 1.2 s total exposure time, at different time points in the relaxation process. Coupled with in crystallo UV-Vis absorption spectroscopy, this RT-MX approach allowed the authors to follow the relaxation of the photoadduct, a thio-ether covalent bond between the chromophore and a cysteine residue. Unexpectedly, the return of the chromophore to its spectroscopic ground state is followed by medium-scale protein rearrangements that trigger a crystal phase transition and hinder the full recovery of the structural ground state of the protein. In addition to suggesting a hitherto unexpected role of a conserved tryptophan residue in the regulation of the photocycle of LOV2, this work provides a basis for performing routine time-resolved protein crystallography experiments at synchrotrons for phenomena occurring on the second-to-hour timescale., (© Sylvain Aumonier et al. 2022.)

Small-angle X-ray scattering (SAXS) is increasingly used to extract structural information from a multitude of soft-matter and biological systems in aqueous solution, including polymers, detergents, lipids, colloids, proteins and RNA/DNA. When SAXS data are recorded at multiple contrasts, i.e. at different electron densities of the solvent, the internal electron-density profile of solubilized molecular systems can be probed. However, contrast-variation SAXS has been limited by the range of electron densities available by conventional agents such as sugars, glycerol and salt, and by the fact that many soft-matter and biological systems are modified in their presence. Here we present a pioneering SAXS contrast-variation study on DDM ( n -do-decyl-β-d-malto-pyran-oside) micelles by using two highly electron-rich contrast agents from biomedical imaging which belong to the families of gadolinium-based and iodinated molecules. The two agents, Gd-HPDO3A and iohexol, were allowed to attain modifications of the solvent electron density that are 50 to 100% higher than those obtained for sucrose, and are located between the electron densities of proteins and RNA/DNA. In the case of Gd-HPDO3A, an analysis of the internal micellar structure was possible and compared with results obtained with sucrose. In conclusion, medical contrast agents represent a promising class of molecules for SAXS contrast-variation experiments with potential appli-cations for numerous soft-matter and biological systems, including membrane proteins and protein-RNA/DNA complexes.

Determining optimal conditions for the production of well diffracting crystals is a key step in every biocrystallography project. Here, a microfluidic device is described that enables the production of crystals by counter-diffusion and their direct on-chip analysis by serial crystallography at room temperature. Nine 'non-model' and diverse biomacromolecules, including seven soluble proteins, a membrane protein and an RNA duplex, were crystallized and treated on-chip with a variety of standard techniques including micro-seeding, crystal soaking with ligands and crystal detection by fluorescence. Furthermore, the crystal structures of four proteins and an RNA were determined based on serial data collected on four synchrotron beamlines, demonstrating the general applicability of this multipurpose chip concept.

Synthetic macrocycles such as calixarenes and cucurbiturils are increasingly applied as mediators of protein assembly and crystallization. The macrocycle can facilitate assembly by providing a surface on which two or more proteins bind simultaneously. This work explores the capacity of the sulfonato-calix[ n ]arene (sclx n ) series to effect crystallization of PAF, a small, cationic antifungal protein. Co-crystallization with sclx 4 , sclx 6 or sclx 8 led to high-resolution crystal structures. In the absence of sclx n , diffraction-quality crystals of PAF were not obtained. Interestingly, all three sclx n were bound to a similar patch on PAF. The largest and most flexible variant, sclx 8 , yielded a dimer of PAF. Complex formation was evident in solution via NMR and ITC experiments, showing more pronounced effects with increasing macrocycle size. In agreement with the crystal structure, the ITC data suggested that sclx 8 acts as a bidentate ligand. The contributions of calixarene size/conformation to protein recognition and assembly are discussed. Finally, it is suggested that the conserved binding site for anionic calixarenes implicates this region of PAF in membrane binding, which is a prerequisite for antifungal activity.