Kinetics of two-step nucleation in protein crystallization studied by real-time SANS

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.

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“…The data correction and absolute intensity calibration were performed using the software LAMP . Data can be accessed via the DOI given in ref .…”

Section: Methodsmentioning

confidence: 99%

Protein Crystallization in the Presence of a Metastable Liquid–Liquid Phase Separation

Maier

Zocher

Sauter

et al. 2020

Crystal Growth & Design

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Evolution of the structure and dynamics of bovine serum albumin induced by thermal denaturation

Matsarskaia

Bühl

Beck

et al. 2020

Phys. Chem. Chem. Phys.

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Kinetics of two-step nucleation in protein crystallization studied by real-time SANS

Cited by 2 publications

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Protein Crystallization in the Presence of a Metastable Liquid–Liquid Phase Separation

Protein Crystallization in the Presence of a Metastable Liquid–Liquid Phase Separation

Evolution of the structure and dynamics of bovine serum albumin induced by thermal denaturation

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