Heterogeneous Crystallization of Proteins: Is it a Prenucleation Clusters Mediated Process?

Abstract

Several studies have shown that heterogeneous agents are able to reduce required starting protein concentration and induction time for crystallization. However, in many cases, the molecular mechanism by which heterogeneous agents promote the nucleation is still unclear, although examples of epitaxial crystallization have been reported. Here, a set of focused crystallization trials, together with a review of the literature data, point out that the process of heterogeneous nucleation can be described by a nonclassical mechanism. We suppose that due to concentration fluctuations, unstable prenucleation aggregates homogeneously form in solution in conditions of low supersaturation, when the chemistry of solution favors this event. Then heterogeneous substrates stabilize prenucleation clusters by nonspecific interactions and trigger the nucleation event. This concept applied to proteins, but also to any colloidal system where the ordered assembly is governed by weak interactions, highlights the importance of the chemistry of the solution as a key parameter to achieve nucleation, even in the presence of heterogeneous nucleants.

LaMer's 1950 model of particle formation: a review and critical analysis of its classical nucleation and fluctuation theory basis, of competing models and mechanisms for phase-changes and particle formation, and then of its application to silver halide, semiconductor, metal, and metal-oxide nanoparticles

“…In modern-day terms, we can recognize Turkevich's creative ''Organizer'' hypothesis as analogous to-and really the first instance of-the currently popular concept of ''prenucleation clusters'', namely the assembly of reaction precursors into large macrostructures with longer-range order that are on-path, kinetically viable nuclei able to undergo facile growth. 72 The concept of prenucleation clusters has been increasingly applied in the literature to the following systems: H 2 SO 4 , 73 CaCO 3 , 72,74 homogeneous crystallizations, Na 2 S 2 O 3 , 76 heterogeneous protein crystallization, 77 glass-type transitions in the solid-state, [78][79][80] and most recently Au(0) n nanoparticles. 81 The allure of the prenucleation cluster concept so noted, one critical issue with prenucleation clusters is if the observed clusters are kinetically on, vs. off, path to the final product.…”

X-ray transparent microfluidic platforms for membrane protein crystallization with microseeds

“…, horse hair, seaweed) which provide nucleation sites for protein crystals despite originating from exogenous materials. 57,58 Further, microseeding as demonstrated on-chip is ideal for applications that require tight control over crystal form and size, such as serial femtosecond crystallography at X-ray free electron lasers, 7,59 and crystallization of proteins for drug delivery. 60,61…”

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

“…The answers to these questions require more work to be done on the characterization of the kinetic signatures associated with different compositions of low-and high-order aggregates (Crespo et al, 2017). Some aspects of nonclassical nucleation mechanisms can be unveiled with the help of advanced microscopic techniques (Fermani et al, 2013). For now, it is important to note the critical role of protein oligomers and metastable clusters in the success of the crystallization step in structural biology pipelines.…”