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We present a systematic study using real-time small-angle neutron scattering (SANS) and optical microscopy to follow the protein crystallization process in the presence of a metastable intermediate...

We study protein crystallization in solutions of human serum albumin (HSA) exhibiting a metastable liquid–liquid phase separation (LLPS) in the presence of trivalent salts. Specifically, we focus on the effects of dense liquid phases (DLPs) on the crystallization pathways. On the basis of the phase diagram, we choose two conditions around the LLPS binodal: one condition is located close to, but outside the LLPS region, resulting in protein clusters, but no macroscopic LLPS. Yet, a surface-enhanced unstable DLP layer is observed at the surface of the cuvette (wetting). The second condition, inside the LLPS binodal, leads to a macroscopic metastable DLP. The crystallization is followed by optical microscopy and small-angle X-ray and neutron scattering (SAXS/SANS) as well as by ultraviolet–visible spectroscopy to explore the role of LLPS. In no case evidence of nucleation inside the DLP is observed. SAXS and SANS show a monotonous growth of the crystals and a decrease of the overall material in the sample. We thus conclude that the existence of a metastable LLPS is not a sufficient condition for a two-step nucleation process. The DLP serves as a reservoir and crystal growth can be described by the Bergeron process, i.e., crystals grow directly into the dilute phase at the expense of the DLP. Furthermore, the crystallographic analysis of the resulting crystals shows that crystals with different morphology grown under different conditions share a similar crystal structure and that the metal ions create two bridging contacts within the unit cell and stabilize it.

A Correction to this paper has been published: https://doi.org/10.1038/s41467-021-21969-9

Coiled-coil protein origami (CCPO) is a modular strategy for the de novo design of polypeptide nanostructures. CCPO folds are defined by the sequential order of concatenated orthogonal coiled-coil (CC) dimer-forming peptides, where a single-chain protein is programmed to fold into a polyhedral cage. Self-assembly of CC-based nanostructures from several chains, similarly as in DNA nanotechnology, could facilitate the design of more complex assemblies and the introduction of functionalities. Here, we show the design of a de novo triangular bipyramid fold comprising 18 CC-forming segments and define the strategy for the two-chain self-assembly of the bipyramidal cage from asymmetric and pseudo-symmetric pre-organised structural modules. In addition, by introducing a protease cleavage site and masking the interfacial CC-forming segments in the two-chain bipyramidal cage, we devise a proteolysis-mediated conformational switch. This strategy could be extended to other modular protein folds, facilitating the construction of dynamic multi-chain CC-based complexes., Coiled-coil protein origami is a strategy for the de novo design of polypeptide nanostructures based on coiled-coil dimer forming peptides, where a single chain protein folds into a polyhedral cage. Here, the authors design a single-chain triangular bipyramid and also demonstrate that the bipyramid can be self-assembled as a heterodimeric complex, comprising pre-defined subunits.

We investigate the transition of the phase separation kinetics from a complete to an arrested liquid–liquid phase separation (LLPS) in mixtures of bovine γ-globulin with polyethylene glycol (PEG). The solutions feature LLPS with upper critical solution temperature phase behavior. At higher PEG concentrations or low temperatures, non-equilibrium, gel-like states are found. The kinetics is followed during off-critical quenches by ultra-small angle X-ray scattering (USAXS) and very-small angle neutron scattering (VSANS). For shallow quenches a kinetics consistent with classical spinodal decomposition is found, with the characteristic length (ξ) growing with time as ξ ∼ t1/3. For deep quenches, ξ grows only very slowly with a growth exponent smaller than 0.05 during the observation time, indicating an arrested phase separation. For intermediate quench depths, a novel growth kinetics featuring a three-stage coarsening is observed, with an initial classical coarsening, a subsequent slowdown of the growth, and a later resumption of coarsening approaching again ξ ∼ t1/3. Samples featuring the three-stage coarsening undergo a temporarily arrested state. We hypothesize that, while intermittent coarsening and collapse might contribute to the temporary nature of the arrested state, migration-coalescence of the minority liquid phase through the majority glassy phase may be the main mechanism underlying this kinetics, which is also consistent with earlier simulation results.

We study the kinetics of the liquid–liquid phase separation (LLPS) and its arrest in protein solutions exhibiting a lower critical solution temperature (LCST) phase behavior using the combination of ultra-small angle X-ray scattering (USAXS) and very-small angle neutron scattering (VSANS). We employ a previously established model system consisting of bovine serum albumin (BSA) solutions with YCl3. We follow the phase transition from sub-second to 104 s upon an off-critical temperature jump. After a temperature jump, the USAXS profiles exhibit a peak that grows in intensity and shifts to lower q values with time. Below 45 °C, the characteristic length scale (ξ) obtained from this scattering peak increases with time with a power of about 1/3 for different sample compositions. This is in good agreement with the theoretical prediction for the intermediate stage of spinodal decomposition where the growth is driven by interface tension. Above 45 °C, ξ follows initially the 1/3 power law growth, then undergoes a significant slowdown, and an arrested state is reached below the denaturation temperature of the protein. This growth kinetics may indicate that the final composition of the protein-rich phase is located close to the high density branch of the LLPS binodal when a kinetically arrested state is reached.