Blocking rapid ice crystal growth through nonbasal plane adsorption of antifreeze proteins

Abstract

Antifreeze proteins (AFPs) are a unique class of proteins that bind to growing ice crystal surfaces and arrest further ice growth. AFPs have gained a large interest for their use in antifreeze formulations for water-based materials, such as foods, waterborne paints, and organ transplants. Instead of commonly used colligative antifreezes such as salts and alcohols, the advantage of using AFPs as an additive is that they do not alter the physicochemical properties of the water-based material. Here, we report the first comprehensive evaluation of thermal hysteresis (TH) and ice recrystallization inhibition (IRI) activity of all major classes of AFPs using cryoscopy, sonocrystallization, and recrystallization assays. The results show that TH activities determined by cryoscopy and sonocrystallization differ markedly, and that TH and IRI activities are not correlated. The absence of a distinct correlation in antifreeze activity points to a mechanistic difference in ice growth inhibition by the different classes of AFPs: blocking fast ice growth requires rapid nonbasal plane adsorption, whereas basal plane adsorption is only relevant at long annealing times and at small undercooling. These findings clearly demonstrate that biomimetic analogs of antifreeze (glyco)proteins should be tailored to the specific requirements of the targeted application.antifreeze protein | thermal hysteresis | ice recrystallization inhibition

Rating antifreeze proteins: Not a breeze

“…After all, TH occurs in the presence of AFPs, irrespective of the particular freezing stimulus and the temperature regime used in the experiment, and it is thus expected that ζ cryo and ζ sono would not differ significantly from one another. By studying six different AFPs that belong to all major AFP families, Olijve et al (10) demonstrate that no such statistically significant correlation exists between the two quantities (Fig. 1B).…”

“…Indeed, neither ssAFP1 ( wfAFP1 (18) are capable of binding to the primary prismatic plane, and they tend to have the two lowest ζ sono values among the AFPs considered in ref. 10. The irrelevance of affinity to basal plane can be clearly seen by comparing rQAE (a recombinant variant of AFP-III) and MPAFP, which both have very similar ζ sono values, despite the latter being incapable of binding to the basal plane.…”

“…The most widely used metric for characterizing the antifreeze activity of an AFP is a quantity known as thermal hysteresis (TH) activity, which is typically determined from nanoliter cryoscopy experiments. In PNAS, Olijve et al (10) demonstrate that the notion of the antifreeze potency of an AFP is complex and process-dependent, and using a single quantity such as TH activity measured from a particular assay is inadequate in predicting the performance of an AFP under different temperature regimes and processing conditions.…”

“…At low AFP concentration, ΔT scales linearly with ffiffiffiffi C p , with C the molar concentration of the AFP under consideration. To correct for the concentration dependence of TH, Olijve et al (10) use the slope of the ΔT vs. ffiffiffiffi C p curve as a proxy for TH activity, a quantity denoted by ζ in this commentary.…”

“…These findings reveal the out-of-equilibrium and pathdependent nature of ζ, and the inadequacy of ζ determined from nanoliter cryoscopy for predicting the TH behavior of the AFP in alternative assays and operating conditions. To explain the molecular origin of this behavior, Olijve et al (10) allude to the fact that different AFPs can bind to different crystallographic planes of ice. The four major crystallographic planes of hexagonal ice (the thermodynamically stable form of ice under ambient pressures) are depicted in Fig.…”