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The function of biomolecules is tightly linked to their structure, and changes therein. Time‐resolved X‐ray solution scattering has proven a powerful technique for interrogating structural changes and signal transduction in photoreceptor proteins. However, these only represent a small fraction of the biological macromolecules of interest. More recently, laser‐induced temperature jumps have been introduced as a more general means of initiating structural changes in biomolecules. Here we present the development of a setup for millisecond time‐resolved X‐ray solution scattering experiments at the CoSAXS beamline, primarily using infrared laser light to trigger a temperature increase, and structural changes. We present results that highlight the characteristics of this setup along with data showing structural changes in lysozyme caused by a temperature jump. Further developments and applications of the setup are also discussed. A new experimental setup for millisecond time‐resolved X‐ray solution scattering has been developed and commissioned at the CoSAXS beamline at the MAX IV synchrotron. Results from T‐jump experiments induced by infrared laser pulses in lysozyme in solution are presented for validation.

Beamline B21 at the Diamond Light Source synchrotron in the UK is a small‐angle X‐ray scattering (SAXS) beamline that specializes in high‐throughput measurements via automated sample delivery systems. A system has been developed whereby a sample can be illuminated by a focused beam of light coincident with the X‐ray beam. The system is compatible with the highly automated sample delivery system at the beamline and allows a beamline user to select a light source from a broad range of wavelengths across the UV and visible spectrum and to control the timing and duration of the light pulse with respect to the X‐ray exposure of the SAXS measurement. The intensity of the light source has been characterized across the wavelength range enabling experiments where a quantitative measure of dose is important. Finally, the utility of the system is demonstrated via measurement of several light‐responsive samples. A system has been developed for irradiating samples with UV and visible light in situ during small‐angle X‐ray scattering data collection. The system is suitable for studying light‐responsive samples such as photosurfactants, UV‐hardening resins or light‐active proteins.

Transition metal ions have been implicated in modulation the conformational behavior and aggregation of WT α‐synuclein (WT‐αSyn), associated with Parkinson's disease pathology. Nevertheless, the initial structural rearrangements that drive aggregation are not fully understood. Here, we employed time‐resolved small‐angle X‐ray scattering (TR‐SAXS) in a microfluidic setup to investigate the structural dynamics of monomeric WT‐αSyn upon interaction with Mn2+, Fe3+, Cu2+, and Zn2+. Using Guinier analysis, GNOM, and Ensemble Optimization Method (EOM), we resolved distinct, metal‐specific conformational transitions on the sub‐second timescale. Fe3+ induced rapid and sustained compaction of αSyn, while Cu2+ promoted extended and heterogeneous conformations, expanding the C‐terminal domain, and disrupting global folding. In contrast, Mn2+ and Zn2+ led to more gradual, domain‐specific compaction. Fractal dimension analysis and hierarchical clustering further revealed Fe3+ and Zn2+ enriched compaction states, while Cu2+ favored intermediate species potentially linked to early aggregation. These findings highlight how metal ion binding differentially and initially reshape the conformation ensemble of WT‐αSyn, offering mechanistic insight into metal‐induced misfolding pathways relevant to synucleinopathies. Time‐resolved SAXS reveals how physiologically relevant metal ions shape the earliest conformational responses of monomeric α‐synuclein. Fe3+ induces rapid compaction, Cu2+ generates heterogeneous and partially folded ensembles, while Mn2+ and Zn2+ cause only modest changes. The real‐time structural fingerprints provide new insight into how metal dyshomeostasis may steer α‐synuclein toward pathogenic aggregation pathways.

“Protein quake” denotes the dissipation of excess energy across a protein, in response to a local perturbation such as the breaking of a chemical bond or the absorption of a photon. Femtosecond time-resolved small- and wide-angle X-ray scattering (TR-SWAXS) is capable of tracking such ultrafast protein dynamics. However, because the structural interpretation of the experiments is complicated, a molecular picture of protein quakes has remained elusive. In addition, new questions arose from recent TR-SWAXS data that were interpreted as underdamped oscillations of an entire protein, thus challenging the long-standing concept of overdamped global protein dynamics. Based on molecular-dynamics simulations, we present a detailed molecular movie of the protein quake after carbon monoxide (CO) photodissociation in myoglobin. The simulations suggest that the protein quake is characterized by a single pressure peak that propagates anisotropically within 500 fs across the protein and further into the solvent. By computing TR-SWAXS patterns from the simulations, we could interpret features in the reciprocal-space SWAXS signals as specific real-space dynamics, such as CO displacement and pressure wave propagation. Remarkably, we found that the small-angle data primarily detect modulations of the solvent density but not oscillations of the bare protein, thereby reconciling recent TR-SWAXS experiments with the notion of overdamped global protein dynamics.

Mixtures of biological macromolecules are inherently difficult to study using structural methods, as increasing complexity presents new challenges for data analysis. Recently, there has been growing interest in studying evolving mixtures using small-angle X-ray scattering (SAXS) in conjunction with time-resolved, high-throughput or chromatography-coupled setups. Deconvolution and interpretation of the resulting datasets, however, are nontrivial when neither the scattering components nor the way in which they evolve are known a priori . To address this issue, the REGALS method (regularized alternating least squares) is introduced, which incorporates simple expectations about the data as prior knowledge, and utilizes parameterization and regularization to provide robust deconvolution solutions. The restraints used by REGALS are general properties such as smoothness of profiles and maximum dimensions of species, making it well suited for exploring datasets with unknown species. Here, REGALS is applied to the analysis of experimental data from four types of SAXS experiment: anion-exchange (AEX) coupled SAXS, ligand titration, time-resolved mixing and time-resolved temperature jump. Based on its performance with these challenging datasets, it is anticipated that REGALS will be a valuable addition to the SAXS analysis toolkit and enable new experiments. The software is implemented in both MATLAB and Python and is available freely as an open-source software package.

This article presents the recent progress of synchrotron scattering methods for elucidating the self-assembly pathways in dilute surfactant systems. An order of magnitude increases in the source brilliance, together with advanced detectors enable time-resolved structural investigations of relatively low contrast and dilute surfactant solutions in the millisecond range. This is demonstrated by monitoring the structural evolution in mixtures of cationic and anionic surfactants well below their individual critical micelle concentration, upon rapid mixing using a stopped-flow device. Despite the low concentration of surfactants in these samples, well-defined nanostructures were detected within in the mixing time of the stopped-flow device (ca. 2 ms). These transient structures have a more elongated morphology than spherical unilamellar vesicles and they grow over the subsequent seconds. The results demonstrate that the spontaneous self-assembly occurs even in dilute solutions without any pre-existing motifs, which may be relevant in applications where high surfactant concentrations are inappropriate.

Small-angle X-ray scattering (SAXS) is an established technique that provides low-resolution structural information on macromolecular solutions. Recent decades have witnessed significant progress in both experimental facilities and in novel data-analysis approaches, making SAXS a mainstream method for structural biology. The technique is routinely applied to directly reconstruct low-resolution shapes of proteins and to generate atomistic models of macromolecular assemblies using hybrid approaches. Very importantly, SAXS is capable of yielding structural information on systems with size and conformational polydispersity, including highly flexible objects. In addition, utilizing high-flux synchrotron facilities, time-resolved SAXS allows analysis of kinetic processes over time ranges from microseconds to hours. Dedicated bioSAXS beamlines now offer fully automated data-collection and analysis pipelines, where analysis and modelling is conducted on the fly. This enables SAXS to be employed as a high-throughput method to rapidly screen various sample conditions and additives. The growing SAXS user community is supported by developments in data and model archiving and quality criteria. This review illustrates the latest developments in SAXS, in particular highlighting time-resolved applications aimed at flexible and evolving systems.

Soft nanoarchitectures created by biomimetic self-assembly offer unexploited potential for therapeutic drug delivery applications, tissue engineering, and diagnostics. The lipid bilayer building blocks impart biocompatible properties and low toxicity of the resulting nanoassemblies. Our work provides a survey of the recent advances in design and structural studies of functional bicontinuous soft porous nanoarchitectures created from amphiphilic bilayer membrane building blocks. Depending on the packing symmetries and the densities of the curved lipid bilayers, organized in membrane-type nanoparticles, a class of multicompartment nanoobjects involving cubosomes, spongosomes, onion-like liposomes, or vesicles arranged in hierarchical supramolecular architectures, can be obtained. High resolution structural investigations by cryo-transmission electron microscopy microscopy and time-resolved small-angle X-ray scattering (SAXS) have demonstrated that binding and complexation of rigid protein molecules to flexible membrane–vesicle building blocks may generate significant changes in the curvature of the membrane interfaces and may induce formation of bicontinuous cubic nanoarchitectures. Enzyme-mediated cubic nanoarchitecture generation represents another low-energy fabrication method of nanoporous liquid crystalline assemblies. The kinetic pathway of packing ready-to-assemble membrane building blocks (vesicles, nanocubosomes) into nanoarchitectonic vehicles has been revealed by rapid-mixing stopped-flow SAXS experiments.

Advances in time-resolved structural techniques, mainly in macromolecular crystallography and small-angle X-ray scattering (SAXS), allow for a detailed view of the dynamics of biological macromolecules and reactions between binding partners. Of particular promise, are mix-and-inject techniques, which offer a wide range of experimental possibility as microfluidic mixers are used to rapidly combine two species just prior to data collection. Most mix-and-inject approaches rely on diffusive mixers, which have been effectively used within crystallography and SAXS for a variety of systems, but their success is dependent on a specific set of conditions to facilitate fast diffusion for mixing. The use of a new chaotic advection mixer designed for microfluidic applications helps to further broaden the types of systems compatible with time-resolved mixing experiments. The chaotic advection mixer can create ultra-thin, alternating layers of liquid, enabling faster diffusion so that even more slowly diffusing molecules, like proteins or nucleic acids, can achieve fast mixing on timescales relevant to biological reactions. This mixer was first used in UV–vis absorbance and SAXS experiments with systems of a variety of molecular weights, and thus diffusion speeds. Careful effort was also dedicated to making a loop-loading sample-delivery system that consumes as little sample as possible, enabling the study of precious, laboratory-purified samples. The combination of the versatile mixer with low sample consumption opens the door to many new applications for mix-and-inject studies.

This review covers recent studies on the characterization of the dynamics of lipidic nanostructures formed via self-assembly processes. The focus is placed on two main topics: First, an overview of advanced experimental small-angle X-ray scattering (SAXS) setups combined with various sample manipulation techniques including, for instance, stop-flow mixing or rapid temperature-jump perturbation is given. Second, our recent synchrotron SAXS findings on the dynamic structural response of gold nanoparticle-loaded vesicles upon exposure to an ultraviolet light source, the impact of rapidly mixing negatively charged vesicles with calcium ions, and in situ hydration-induced formation of inverted-type liquid-crystalline phases loaded with the local anesthetic bupivacaine are summarized. These in situ time-resolved experiments allow real-time monitoring of the dynamics of the structural changes and the possible formation of intermediate states in the millisecond to second range. The need for investigating self-assembled systems, mainly stimuli-responsive drug nanocarriers, under nonequilibrium conditions is discussed. For pharmaceutically relevant applications, it is essential to combine these investigations with appropriate in vitro and in vivo studies.