Hetero- vs Homogeneous Nucleation of Protein Crystals Discriminated by Supersaturation

In protein crystallization trials there is always some ambiguity in defining if the nucleation event occurred by a homogeneous or a heterogeneous process. One of the reasons comes from the difficulty to compare experiments with or without a heterogeneous nucleant under the same chemical conditions. The outcome is that it is difficult to figure out the values of supersaturation addressing homo- versus heterogeneous nucleation. Recently it has been shown that the crystallization mushroom allows performance of sitting drop vapor diffusion experiments under identical physical-chemical conditions and that functionalized surfaces are favorable heterogeneous substrates for protein crystallization. With the aim to discriminate between nucleation processes, three model proteins, lysozyme, glucose isomerase, and thaumatin, have been crystallized at different starting concentrations, using positively and negatively charged surfaces, in the crystallization mushroom. The results show that (i) the heterogenenous nucleation does not crucially affect the nucleation process under conditions of high supersaturation and that (ii) the nucleating action of the fianctionalized surfaces is mainly related to their superficial charge distribution more than to their absolute charge, according to both classical and colloidal nucleation theories.