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 (T₅₀ = 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.