Simulation Guided Design of a Potentially Hyperactive Ice Nucleating Protein

Ice nucleating proteins (INPs) are among the most potent biological catalysts for heterogeneous ice nucleation, triggering freezing at temperatures near 0 °C. Despite structural insights into their β-helical repeat, the molecular mechanisms governing their nucleation efficiency are not fully understood. Extensive atomistic molecular dynamics (MD) simulations (50 μs total) were used to investigate ice nucleation by a modeled Pseudomonas syringae INP (inaV central repeat domain, residues 221–476). Simulations captured ice formation initiating predominantly at YGS and TxT motifs, followed by propagation into stable crystals. Based on these findings, a modified INP with doubled YGS and TxT motifs was designed. This variant displayed faster nucleation kinetics ( T 50 = 1.9 ± 0.4 μs compared to 3.3 ± 1.8 μs for WT) and ∼28.5% higher ice-like water fractions near key motifs. These findings characterize the YGS motif as a potent water-organizing element and demonstrate the efficacy of MD-driven design in enhancing INP performance for possible energy-saving and biotechnological applications.