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The kinetics of heterogeneous polymerization is determined directly using small-angle X-ray scattering (SAXS). This important advancement is exemplified for the synthesis of sterically-stabilized diblock copolymer nanoparticles by reversible addition-fragmentation chain transfer (RAFT) dispersion polymerization of benzyl methacrylate (BzMA) in mineral oil at 90 °C. The principle of mass balance is invoked to derive a series of equations for the analysis of the resulting time-resolved SAXS patterns. Importantly, there is a continuous change in the X-ray scattering length density for the various components within the reaction mixture. This enables the volume fraction of unreacted BzMA monomer to be calculated at any given time point, which enables the polymerization kinetics to be monitored in situ directly without relying on supplementary characterization techniques. Moreover, SAXS enables the local concentration of both monomer and solvent within the growing swollen nanoparticles to be determined during the polymerization. Data analysis reveals that the instantaneous rate of BzMA polymerization is proportional to the local monomer concentration within the nanoparticles. In principle, this powerful new time-resolved SAXS approach can be applicable to other heterogeneous polymerization formulations.

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Capsid assembly modulators (CAMs) are antiviral molecules that disturb the formation of icosahedral viral capsids, in particular, those of the Hepatitis B virus (HBV). We report an integrated, physics-driven study elucidating quantitatively the effects of two classes of CAMs on the HBV capsid assembly. Time-resolved small-angle X-ray scattering measurements revealed accelerated self-assembly processes that implied the increase of subunit binding energy from 9- up to 18-fold the thermal energy due to CAMs. Cryotransmission electron microscopy images showed that both classes induce various changes in capsid morphology: from a slight elongation, unrecognized in previous work, to a strong deformation with a capsid size more than twice as large. The observed capsid morphologies were closely reproduced in coarse-grained simulations by varying the Föppl-von-Kármán number, thus pointing out the role of CAMs in altering the capsid elastic energy. Our results illuminate the mechanisms of action of CAMs on HBV capsid assembly at high spatiotemporal resolution and may bring perspectives on virus-derived nanocapsules with tunable morphologies.

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Tubulin, an essential cytoskeletal protein, assembles into various morphologies by interacting with an array of cellular factors. One of these factors is the endogenous polyamine spermine, which may promote and stabilize tubulin assemblies. Nevertheless, the assembled structures and their formation pathways are poorly known. Here we show that spermine induced the in vitro assembly of tubulin into several hierarchical architectures based on a tubulin conical-spiral subunit. Using solution X-ray scattering and cryo-TEM, we found that with progressive increase of spermine concentration tubulin dimers assembled into conical-frustum-spirals of increasing length, containing up to three helical turns. The subunits with three helical turns were then assembled into tubules through base-to-top packing and formed antiparallel bundles of tubulin conical-spiral tubules in a distorted hexagonal symmetry. Further increase of the spermine concentration led to inverted tubulin tubules assembled in hexagonal bundles. Time-resolved experiments revealed that tubulin assemblies formed at higher spermine concentrations assembled from intermediates, similar to those formed at low spermine concentrations. These results are distinct from the classical transition between twisted ribbons, helical, and tubular assemblies, and provide insight into the versatile morphologies that tubulin can form. Furthermore, they may contribute to our understanding of the interactions that control the composition and construction of protein-based biomaterials.

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Recent structures of full-length ATP-binding cassette (ABC) transporter MsbA in different states indicate large conformational changes during the reaction cycle that involve transient dimerization of its nucleotide-binding domains (NBDs). However, a detailed molecular understanding of the structural changes and associated kinetics of MsbA upon ATP binding and hydrolysis is still missing. Here, we employed time-resolved small-angle X-ray scattering, initiated by stopped-flow mixing, to investigate the kinetics and accompanying structural changes of NBD dimerization (upon ATP binding) and subsequent dissociation (upon ATP hydrolysis) in the context of isolated NBDs as well as full-length MsbA in lipid nanodiscs. Our data allowed us to structurally characterize the major states involved in the process and determine time constants for NBD dimerization and dissociation. In the full-length protein, these structural transitions occur on much faster time scales, indicating close-proximity effects and structural coupling of the transmembrane domains with the NBDs.

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Self-assembled nanocrystal superlattices have attracted large scientific attention due to their potential technological applications. However, the nucleation and growth mechanisms of superlattice assemblies remain largely unresolved due to experimental difficulties to monitor intermediate states. Here, the self-assembly of colloidal PbS nanocrystals is studied in real time by a combination of controlled solvent evaporation from the bulk solution and in situ small-angle X-ray scattering (SAXS) in transmission geometry. For the first time for the investigated system a hexagonal closed-packed (hcp) superlattice formed in a solvent vapor saturated atmosphere is observed during slow solvent evaporation from a colloidal suspension. The highly ordered hcp superlattice is followed by a transition into the final body-centered cubic superlattice upon complete drying. Additionally, X-ray cross-correlation analysis of Bragg reflections is applied to access information on precursor structures in the assembly process, which is not evident from conventional SAXS analysis. The detailed evolution of the crystal structure with time provides key results for understanding the assembly mechanism and the role of ligand-solvent interactions, which is important both for fundamental research and for fabrication of superlattices with desired properties.

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Viral capsids derived from an icosahedral plant virus widely used in physical and nanotechnological investigations were fully dissociated into dimers by a rapid change of pH. The process was probed in vitro at high spatiotemporal resolution by time-resolved small-angle X-ray scattering using a high brilliance synchrotron source. A powerful custom-made global fitting algorithm allowed us to reconstruct the most likely pathway parametrized by a set of stoichiometric coefficients and to determine the shape of two successive intermediates by ab initio calculations. None of these two unexpected intermediates was previously identified in self-assembly experiments, which suggests that the disassembly pathway is not a mirror image of the assembly pathway. These findings shed new light on the mechanisms and the reversibility of the assembly/disassembly of natural and synthetic virus-based systems. They also demonstrate that both the structure and dynamics of an increasing number of intermediate species become accessible to experiments.

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