The cover image represents protein-rich clusters in insulin solution, where they likely serve as precursors to crystal nucleation. Insulin crystallization is a part of insulin biosynthesis in the mammalian pancreas and an essential step in the manufacture of diabetes medicine. The image was taken by oblique illumination dark-field microscopy and artificial coloring was applied for artistic effect.
Protein-rich clusters are of interest as nucleation precursors in protein crystallization, which is a part of industrial processes and laboratory procedures and is notoriously difficult. Oftentimes crystals never form but instead amorphous aggregates, fibrils, dense liquids, and other undesirables appear. The main kinetic impediment to protein nucleation appears to be the relatively large surface free energy g of the crystal-solution interface. In combination with the large size of the protein molecules, the high g leads to a high free energy barrier for nucleation. In turn, this high barrier imposes high supersaturations at which nucleation may occur, thus expediting the formation of undesired solid phases. To understand how proteins work around the high surface free energy problem, the two-step mechanism of nucleation was put forth. According to this mechanism, crystal nuclei assemble within preexisting protein-rich clusters rather than from molecules in the dilute solution. Owing to the high protein concentration in the clusters, the surface free energy of a crystalline nucleus emerging in them drops off by up to two orders of magnitude from g of a nucleus forming in the solution. Thus, the protein-rich clusters emerge as crucial prerequisites for protein crystal nucleation.
The protein-rich clusters are much larger than the prediction of a colloid clustering scenario, which assumes structurally intact molecules and evaluates the balance of attractive and repulsive forces between them. Our group put forth an alternative kinetic mechanism, according to which the clusters consist of a concentrated mixture of protein complexes and monomers. Our paper in the November 3 issue of Biophysical Journal presents decisive evidence in favor of a kinetic mechanism. We use the highly basic protein lysozyme at nearly neutral and lower pH as a model and explore the response of the cluster population to the electrostatic forces, which govern numerous biophysical phenomena, including crystallization and fibrillization. The results demonstrate that the Coulomb forces that govern aggregation in biological systems and many other phenomena in nature do not affect the cluster size. In combination with other cluster behaviors, this response demonstrates that the mesoscopic clusters represent a novel class of protein condensate. The clusters form by a unique mechanism, which includes the accumulation of transient protein oligomers that are linked by hydrophobic bonds between the peptide backbones exposed to the solvent after partial protein unfolding. Our findings indicate that fine-tuning of the intra- and inter-molecular water-structuring interactions may be an essential tool to control the cluster population and in this way enhance or suppress protein crystallization and fibrillization.
– Maria Vorontsova, Ho Yin Chan, Vassiliy Lubchenko, Peter Vekilov