Ice nucleation in aqueous solutions of short- and long-chain poly(vinyl alcohol) studied with a droplet microfluidics setup.

Poly(vinyl alcohol) (PVA) has ice binding and ice nucleating properties. Here, we explore the dependence of the molecular size of PVA on its ice nucleation activity. For this purpose, we studied ice nucleation in aqueous solutions of PVA samples with molar masses ranging from 370 to 145 000 g mol-1, with a particular focus on oligomer samples with low molar mass. The experiments employed a novel microfluidic setup that is a follow-up on the previous WeIzmann Supercooled Droplets Observation on a Microarray (WISDOM) design by Reicher et al. The modified setup introduced and characterized here, termed nanoliter Bielefeld Ice Nucleation ARraY (nanoBINARY), uses droplet microfluidics with droplets (96 ± 4) µm in diameter and a fluorinated continuous oil phase and surfactant. A comparison of homogeneous and heterogeneous ice nucleation data obtained with nanoBINARY to those obtained with WISDOM shows very good agreement, underpinning its ability to study low-temperature ice nucleators as well as homogeneous ice nucleation due to the low background of impurities. The experiments on aqueous PVA solutions revealed that the ice nucleation activity of shorter PVA chains strongly decreases with a decrease in molar mass. While the cumulative number of ice nucleating sites per mass nm of polymers with different molar masses is the same, it becomes smaller for oligomers and completely vanishes for dimer and monomer representatives such as 1,3-butanediol, propan-2-ol, and ethanol, most likely because these molecules become too small to effectively stabilize the critical ice embryo. Overall, our results are consistent with PVA polymers and oligomers acting as heterogeneous ice nucleators.

Water-organizing motif continuity is critical for potent ice nucleation protein activity.

Bacterial ice nucleation proteins (INPs) can cause frost damage to plants by nucleating ice formation at high sub-zero temperatures. Modeling of Pseudomonas borealis INP by AlphaFold suggests that the central domain of 65 tandem sixteen-residue repeats forms a beta-solenoid with arrays of outward-pointing threonines and tyrosines, which may organize water molecules into an ice-like pattern. Here we report that mutating some of these residues in a central segment of P. borealis INP, expressed in Escherichia coli, decreases ice nucleation activity more than the section's deletion. Insertion of a bulky domain has the same effect, indicating that the continuity of the water-organizing repeats is critical for optimal activity. The ~10 C-terminal coils differ from the other 55 coils in being more basic and lacking water-organizing motifs; deletion of this region eliminates INP activity. We show through sequence modifications how arrays of conserved motifs form the large ice-nucleating surface required for potency.

Contrasting Behavior of Antifreeze Proteins: Ice Growth Inhibitors and Ice Nucleation Promoters.

Several types of natural molecules interact specifically with ice crystals. Small antifreeze proteins (AFPs) adsorb to particular facets of ice crystals, thus inhibiting their growth, whereas larger ice-nucleating proteins (INPs) can trigger the formation of new ice crystals at temperatures much higher than the homogeneous ice nucleation temperature of pure water. It has been proposed that both types of proteins interact similarly with ice and that, in principle, they may be able to exhibit both functions. Here we investigated two naturally occurring antifreeze proteins, one from fish, type-III AFP, and one from beetles, TmAFP. We show that in addition to ice growth inhibition, both can also trigger ice nucleation above the homogeneous freezing temperature, providing unambiguous experimental proof for their contrasting behavior. Our analysis suggests that the predominant difference between AFPs and INPs is their molecular size, which is a very good predictor of their ice nucleation temperature.