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

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.