Effects of Ice Nucleation Protein Repeat Number and Oligomerization Level on Ice Nucleation Activity

Ice nucleation active bacteria have attracted particular attention due to their unique ability to produce specific ice nucleation proteins (INpros), which are the most efficient ice nuclei known as they induce nucleation at temperatures close to 0°C. Our model bacterium Pseudomonas syringae strain R10.79 produced INpros containing 67 tandem repeats, forming the proposed ice-binding surface. To understand the role of the INpro repeats as well as the role of intermolecular interactions between INpros for their ice nucleation behavior, we produced a truncated version of the protein with only 16 tandem repeats (INpro_16R). The purified INpro_16R produced oligomers of varying sizes. Immersion freezing ice nucleation behavior of purified INpro_16R was characterized by droplet-freezing assays and in the Leipzig Aerosol Cloud Interaction Simulator. Predominant INpro_16R oligomers introduced into Leipzig Aerosol Cloud Interaction Simulator as single particles with diameters of 50 nm or 70 nm were ice nucleation active at temperatures of -26°C and -24°C, respectively. These are much lower temperatures compared to that of intact INpros (-12°C). The data clearly indicated that the number of repeats determines the ice nucleation temperature. In addition, ice nucleation between -9°C and -10°C, comparable to the activity of intact INpro, was caused by higher-order INpro_16R oligomers. This supported previous observations that INpro oligomerization increases the ice-binding surface, thereby affecting ice nucleation activity. In conclusion, both repeat number and oligomerization contribute in a seemingly independent manner to the nucleation mechanism of INpros.