Ran Drori

Microfluidic experiments reveal that antifreeze proteins bound to ice crystals suffice to prevent their growth

Antifreeze proteins (AFPs) are a subset of ice-binding proteins that control ice crystal growth. They have potential for the cryopreservation of cells, tissues, and organs, as well as for production and storage of food and protection of crops from frost. However, the detailed mechanism of action of AFPs is still unclear. Specifically, there is controversy regarding reversibility of binding of AFPs to crystal surfaces. The experimentally observed dependence of activity of AFPs on their concentration in solution appears to indicate that the binding is reversible. Here, by a series of experiments in temperature-controlled microfluidic devices, where the medium surrounding ice crystals can be exchanged, we show that the binding of hyperactive Tenebrio molitor AFP to ice crystals is practically irreversible and that surface-bound AFPs are sufficient to inhibit ice crystal growth even in solutions depleted of AFPs. These findings rule out theories of AFP activity relying on the presence of unbound protein molecules.

Ice-binding proteins that accumulate on different ice crystal planes produce distinct thermal hysteresis dynamics.

Ice-binding proteins that aid the survival of freeze-avoiding, cold-adapted organisms by inhibiting the growth of endogenous ice crystals are called antifreeze proteins (AFPs). The binding of AFPs to ice causes a separation between the melting point and the freezing point of the ice crystal (thermal hysteresis, TH). TH produced by hyperactive AFPs is an order of magnitude higher than that produced by a typical fish AFP. The basis for this difference in activity remains unclear. Here, we have compared the time dependence of TH activity for both hyperactive and moderately active AFPs using a custom-made nanolitre osmometer and a novel microfluidics system. We found that the TH activities of hyperactive AFPs were time-dependent, and that the TH activity of a moderate AFP was almost insensitive to time. Fluorescence microscopy measurement revealed that despite their higher TH activity, hyperactive AFPs from two insects (moth and beetle) took far longer to accumulate on the ice surface than did a moderately active fish AFP. An ice-binding protein from a bacterium that functions as an ice adhesin rather than as an antifreeze had intermediate TH properties. Nevertheless, the accumulation of this ice adhesion protein and the two hyperactive AFPs on the basal plane of ice is distinct and extensive, but not detectable for moderately active AFPs. Basal ice plane binding is the distinguishing feature of antifreeze hyperactivity, which is not strictly needed in fish that require only approximately 1°C of TH. Here, we found a correlation between the accumulation kinetics of the hyperactive AFP at the basal plane and the time sensitivity of the measured TH.

Experimental correlation between thermal hysteresis activity and the distance between antifreeze proteins on an ice surface

Antifreeze proteins (AFPs) aid the survival of cold-adapted organisms by inhibiting the growth of ice crystals in the organism. The binding of AFPs to ice separates the melting point from the freezing point of the ice crystal (thermal hysteresis, TH). Although AFPs were discovered more than 40 years ago, the mechanism by which they inhibit ice growth remains unclear. The distance between surface-bound AFPs is thought to correlate directly with the TH activity; however, this correlation has never been experimentally established. A novel microfluidics system was used here to obtain ice crystals covered with GFP-tagged AFPs in an AFP-free solution. This method permits calculation of the surface density of bound AFPs. Fluorescence intensity analysis revealed that the distance between ∼3 nm-long AFPs on the ice surface was 7–35 nm, depending on the AFP solution concentration and time of its exposure to ice. A direct correlation between these distances and the measured TH activity was found for a representative insect AFP, but not for a typical fish AFP. Insect AFPs accumulate over multiple ice crystal planes, especially the basal plane. Fish AFPs, which cannot bind to the basal plane, change the shape of the crystal to minimize the basal plane area. Thus, we postulate that the surface density of fish AFPs on the prism plane is not directly indicative of the TH activity, which ends when ice grows out of the basal plane and is a function of the basal plane area. These results significantly contribute to our understanding of the AFP mechanism and will be helpful in applying these proteins in different fields.

Dendrimer-Linked Antifreeze Proteins Have Superior Activity and Thermal Recovery.

By binding to ice, antifreeze proteins (AFPs) depress the freezing point of a solution and inhibit ice recrystallization if freezing does occur. Previous work showed that the activity of an AFP was incrementally increased by fusing it to another protein. Even larger increases in activity were achieved by doubling the number of ice-binding sites by dimerization. Here, we have combined the two strategies by linking multiple outward-facing AFPs to a dendrimer to significantly increase both the size of the molecule and the number of ice-binding sites. Using a heterobifunctional cross-linker, we attached between 6 and 11 type III AFPs to a second-generation polyamidoamine (G2-PAMAM) dendrimer with 16 reactive termini. This heterogeneous sample of dendrimer-linked type III constructs showed a greater than 4-fold increase in freezing point depression over that of monomeric type III AFP. This multimerized AFP was particularly effective at ice recrystallization inhibition activity, likely because it can simultaneously bind multiple ice surfaces. Additionally, attachment to the dendrimer has afforded the AFP superior recovery from heat denaturation. Linking AFPs together via polymers can generate novel reagents for controlling ice growth and recrystallization.