Freezing-induced phase separation and spatial microheterogeneity in protein solutions

Amid decades of research, the basic mechanisms of lyo-/cryostabilization of proteins and more complex organisms have not yet been fully established. One major bottleneck is the inability to probe into and control the molecular level interactions. The molecular interactions are responsible for the significant differences in the outcome of the preservation processes.¹ In this communication, we have utilized confocal Raman microspectroscopy to quantify the freezing-induced microheterogeneity and phase separation (solid and liquid) in a frozen solution composed of a model protein (lysozyme) and a lyo-/cryoprotectant (trehalose), which experienced different degrees of supercooling. Detailed quantitative spectral analysis was performed across the ice, the freeze-concentrated liquid (FCL) phases, and the interface region between them. It was established that the characteristics of the microstructures observed after freezing depended not only on the concentration of trehalose in the solution but also on the degree of supercooling. It was shown that, when samples were frozen after high supercooling, small amounts of lysozyme and trehalose were occluded in the ice phase. Lysozyme preserved its native-like secondary structure in the FCL region but was denatured in the ice phase. Also, it was observed that induction of freezing after a high degree of supercooling of high trehalose concentrations resulted in aggregation of the sugar and the protein.