Electron microscopy and calorimetry of proteins in supercooled water

Some of the best nucleating agents in nature are ice-nucleating proteins, which boost ice growth better than any other material. They can induce immersion freezing of supercooled water only a few degrees below 0 °C. An open question is whether this ability also extends to the deposition mode, i.e., to water vapor. In this work, we used three proteins, apoferritin, InaZ (ice nucleation active protein Z), and myoglobin, of which the first two are classified as ice-nucleating proteins for the immersion freezing mode. We studied the ice nucleation ability of these proteins by differential scanning calorimetry (immersion freezing) and by environmental scanning electron microscopy (deposition freezing). Our data show that InaZ crystallizes water directly from the vapor phase, while apoferritin first condenses water in the supercooled state, and subsequently crystallizes it, just as myoglobin, which is unable to nucleate ice.