Simulation Guided Design of a Potentially Hyperactive Ice Nucleating Protein

Area of Science:

• Biophysics
• Materials Science
• Protein Engineering

Background:

• Ice nucleating proteins (INPs) are highly efficient biological catalysts for ice formation near 0 °C.
• The precise molecular mechanisms governing INP nucleation efficiency, despite known β-helical repeats, remain incompletely understood.

Purpose of the Study:

• To investigate ice nucleation mechanisms by a modeled *Pseudomonas syringae* INP using atomistic molecular dynamics simulations.
• To identify key motifs responsible for ice nucleation and guide the design of enhanced INPs.

Main Methods:

• Conducted extensive atomistic molecular dynamics (MD) simulations totaling 50 μs on a *Pseudomonas syringae* INP (inaV central repeat domain).
• Analyzed simulation trajectories to pinpoint initiation sites and propagation of ice formation.
• Designed and simulated a modified INP variant with doubled YGS and TxT motifs.

Main Results:

• Ice nucleation was observed to initiate predominantly at YGS and TxT motifs, facilitating stable crystal formation.
• The modified INP variant exhibited significantly faster nucleation kinetics ( *T*₅₀ = 1.9 ± 0.4 μs) compared to wild-type (WT) (3.3 ± 1.8 μs).
• The enhanced INP showed approximately 28.5% higher ice-like water fractions near the key YGS and TxT motifs.

Conclusions:

• The YGS motif is identified as a critical element for organizing water molecules during ice nucleation.
• Molecular dynamics-driven protein design is effective in enhancing INP performance.
• Optimized INPs hold potential for energy-saving and biotechnological applications.