The nucleation of protein crystals as a race against time with on- and off-pathways

Abstract

High supersaturation levels are a necessary but insufficient condition for the crystallization of purified proteins. Unlike most small molecules, proteins can take diverse aggregation pathways that make the outcome of crystallization assays quite unpredictable. Here, dynamic light scattering and optical microscopy were used to show that the nucleation of lysozyme crystals is preceded by an initial step of protein oligomerization and by the progressive formation of metastable clusters. Because these steps deplete the concentration of soluble monomers, the probability of obtaining protein crystals decreases as time progresses. Stochastic variations of the induction time are thus amplified to a point where fast crystallization can coexist with unyielding regimes in the same conditions. With an initial hydrodynamic radius of $100 nm, the metastable clusters also promote the formation of protein crystals through a mechanism of heterogeneous nucleation. Crystal growth (on-pathway) takes place in parallel with cluster growth (off-pathway). The Janus-faced influence of the mesoscopic clusters is beneficial when it accelerates the formation of the first precrystalline nuclei and is detrimental as it depletes the solution of protein ready to crystallize. Choosing the right balance between the two effects is critical for determining the success of protein crystallization trials. The results presented here suggest that a mild oligomerization degree promotes the formation of a small number of metastable clusters which then catalyze the nucleation of well differentiated crystals.

Probing the Occurrence of Soluble Oligomers through Amyloid Aggregation Scaling Laws

“…Pre-filtration of the insulin solution using 0.22 μm syringe filters efficiently removed these particles (Figure S2), but it also delayed the onset of the fibrillation process until a point where the kinetic measurements of Foderà et al [26] could not be reproduced anymore. Therefore, pre-assembled protein clusters act as important heterogeneous nucleation centers without which the rapid formation of ordered aggregates is compromised [37,38,39]. The low concentration of the insulin clusters (fraction of total protein <10−10 estimated from the initial PSDs) is high enough to conceal the initial progress of fibril sizes expected to start at R*=5.7 nm and not from R¯h values greater than 100 nm (compare dashed lines and experimental values in Figure 4D).…”

The Study of the Mechanism of Protein Crystallization in Space by Using Microchannel to Simulate Microgravity Environment

“…The effect of convection on the protein growth mechanism may be manifold. Novel investigations suggest that the protein molecules can form oligomers already in the crystallization solution [63], rather than entering the crystal lattice one-by-one. Ferreira et al [63] thought that the mesoscopic clusters are beneficial when they accelerate the formation of the first precrystalline nuclei and are detrimental as they deplete the solution of protein ready to crystallize.…”

“…Novel investigations suggest that the protein molecules can form oligomers already in the crystallization solution [63], rather than entering the crystal lattice one-by-one. Ferreira et al [63] thought that the mesoscopic clusters are beneficial when they accelerate the formation of the first precrystalline nuclei and are detrimental as they deplete the solution of protein ready to crystallize. Nadarjah et al [64] thought that the growth of a protein crystal was a two-step process: aggregate growth units are first formed in the bulk solution by stronger intermolecular bonds and then attached to the crystal face by weaker bonds.…”

Classical and Nonclassical Nucleation Mechanisms of Insulin Crystals

“…Sleutel and van Driessche (2014) 5 showed evidence that clusters are liquid-like and metastable concerning the emerging crystalline phase (e.g., lysozyme and insulin). Ferreira et al (2017) 10 demonstrated the existence of clusters that do not give rise to crystals and remain in solution (lysozyme). Kaissaratos et al (2021) 8 also stated that two-step nucleation is a phase transformation over the classical pathway due to a lower surface-free energy barrier.…”