How Do Size and Aggregation of Ice-binding Proteins Control their Ice Nucleation Efficiency

Organisms that thrive at cold temperatures have evolved ice-binding proteins to manage the nucleation and growth of ice. Bacterial ice-nucleating proteins (INP) and insect hyperactive antifreeze proteins (AFP) bind ice through the same amino acid motifs, despite their opposite functions. AFPs are generally small, while INPs are long and aggregate in the cell membrane. It is not yet understood to which extent the size and aggregation determine the temperature Thet at which proteins nucleate ice. Here we address this question using molecular simulations and nucleation theory. The simulations indicate that the 2.5 nm long Tm AFP nucleates ice at 2±1 ° C above the homogeneous nucleation temperature. Addition of ice-binding loops to Tm AFP increases Thet until the length of the binding-site becomes ~4 times its width, beyond which Thet plateaus. We calculate that the INP of Ps. syringae, Ps INP, reaches its maximum Thet = -26 ° C when its binding site has 16 ice-binding loops, in excellent agreement with Thet = -25 ±1 ° C measured for an engineered 16-loop fragment of Ps INP. To further increase Thet , the proteins must aggregate. We predict Thet per number of Ps INP in the aggregate, and conclude that assemblies with 34 INP already reach Thet = -2 ° C characteristic of this bacterium. Interestingly, we find that Thet of aggregates is a non-monotonic and strongly varying function of the distance between proteins. We conclude that to achieve maximum freezing efficiency, bacteria must exert exquisite, sub-angstrom control of the distance between INP in their membrane.