Interfacial Water Ordering Is Insufficient to Explain Ice-Nucleating Protein Activity

Area of Science:

• Biochemistry
• Microbiology
• Physical Chemistry

Background:

• Ice-nucleating proteins (INPs) from bacteria are highly efficient at inducing water crystallization near 0 °C.
• The precise mechanism behind INPs' potent ice nucleation activity remains incompletely understood.
• Understanding INP function is key to applications in cryopreservation and weather modification.

Purpose of the Study:

• To elucidate the structural dynamics of Pseudomonas syringae INPs in solution.
• To determine the relationship between INP structure, thermal stability, and ice nucleation activity.
• To investigate the role of protein structure versus water ordering in INP function.

Main Methods:

• Solution structure analysis of INPs using spectroscopic techniques.
• Thermal stress experiments to assess protein stability and activity loss.
• Sum-frequency generation (SFG) spectroscopy to probe interfacial water structure.

Main Results:

• INPs exhibit a stable solution structure that does not change significantly upon cooling.
• Heating INPs above approximately 55 °C causes irreversible structural changes and loss of ice nucleation activity.
• Both active and heat-inactivated INPs induce similar ordering of interfacial water molecules during cooling.

Conclusions:

• Increased water ordering at the protein interface is insufficient to explain high ice nucleation activity.
• The integrity of the three-dimensional protein structure is essential for bacterial ice nucleation.
• Bacterial ice nucleation likely relies on supramolecular interactions mediated by intact INPs.