How Size and Aggregation of Ice-Binding Proteins Control Their Ice Nucleation Efficiency

Area of Science:

• Biophysics
• Materials Science
• Microbiology

Background:

• Organisms in cold environments utilize ice-binding proteins to regulate ice formation.
• Bacterial ice-nucleating proteins (INP) are membrane-bound aggregates, while insect antifreeze proteins (AFP) are soluble and small.
• Warmer ice nucleation temperatures correlate with larger ice-binding proteins and aggregates, but quantitative understanding is lacking.

Purpose of the Study:

• To quantitatively determine how the size and aggregation of ice-binding proteins influence ice nucleation temperature (T_het).
• To compare the ice nucleation efficiency of bacterial INP with insect AFP.
• To predict the T_het of bacterial INP aggregates using nucleation theory.

Main Methods:

• Molecular simulations were employed to study ice nucleation by antifreeze protein TmAFP.
• Classical nucleation theory was adapted to determine T_het for finite-sized surfaces.
• The theoretical model was validated against simulation results and then used to predict T_het for bacterial INP aggregates.

Main Results:

• The antifreeze protein TmAFP nucleated ice at 2 ± 1 °C above the homogeneous nucleation temperature.
• Adding ice-binding loops to TmAFP increased T_het but did not match bacterial INP efficiency.
• Bacterial INP aggregates of 34 proteins achieved the characteristic T_het of -2 °C.
• T_het exhibited a nonmonotonic dependence on the distance between INP in aggregates.

Conclusions:

• Protein size and aggregation significantly impact ice nucleation temperature.
• Bacteria require precise subangstrom control over INP spacing in membranes for optimal freezing efficiency.
• This study provides a quantitative framework for understanding ice nucleation by protein assemblies.