144 Kinetics of hyperactive and moderate antifreeze proteins

Abstract

Ice binding proteins (IBPs) protect cold-adapted organisms from freeze injuries by inhibiting the growth of endogenous ice crystals. IBPs have been found in fish, insects, plants, fungi and bacteria, and hold great potential in different fields such as the food industry, medicine and agriculture. IBPs include antifreeze proteins (AFPs), which adsorb to the surface of ice crystals and lower the temperature at which ice crystals grow, thereby creating a gap (thermal hysteresis [TH]) between the melting point and the non-equilibrium freezing point, within which ice growth is arrested. The accepted model explaining the mechanism of action of AFPs is the adsorption–inhibition model (Raymond and DeVries, 1977). Although this model requires irreversible binding of AFP to ice, there is still an ongoing debate over whether AFPs adsorb to ice irreversibly; furthermore, the time-dependence of this process is still unclear. Our results on the kinetics of AFP binding show that for a hyperactive AFP from Tenebrio molitor (TmAFP), TH activity increased with time by 3- to 10-fold for all of the concentrations measured (1–40 μM). In an AFP from spruce budworm ( Choristoneura fumiferana ) (sbwAFP) the increase of TH activity with time was the highest, reaching a 43-fold increase. Using a novel microfluidic system (Celik, Drori et al., 2013) to investigate the binding mechanism of AFPs to ice, we exposed an ice crystal to AFP molecules, and then exchanged the bathing solution with AFP-free buffer. Assuming irreversible binding, upon exchange of the AFP solution, the crystal must remain covered with AFPs. Our results with a TmAFP-GFP solution show that after the removal of the AFP solution, TH activity was similar to that before the exchange, and the crystal remains covered with TmAFP-GFP (Celik, Drori et al., 2013). Similar to the exchange of TmAFP-GFP solution, after the removal of an AFPIII-GFP solution, bound AFP were sufficient to inhibit ice growth in a supercooled solution (0.11–0.23 °C). However, the ice growth inhibition of AFPIII (which do not bind to the basal plane of the crystal) is dependent on the crystal shape; if the bipyramidal shape is complete, the basal plane is minimal and ice growth is suppressed. Our microfluidic system also enables us to measure and calculate the spacing between AFP molecules on the ice surface. We found that depending on solution concentration and exposure time of the crystal, the distance between AFPs is 5–30 nm (note that the dimensions of AFPs are about 3 nm 2 ), and this value has a direct correlation to the TH activity. Our results suggest that AFPs accumulate on the ice surface, and that any desorption rate must be much slower than the adsorption rate. These findings reveal new insights about the mechanism of action of AFPs, and assist in better understanding how to manipulate these proteins for our benefit. Source of funding: This research was funded by Israel Science Foundation, The European Research Council, and the Canadian Institutes of Health Research. Conflict of interest: None declared. drori.ran@gmail.com