COMMENTARY COMMENTARY

Rating antifreeze proteins: Not a breeze Amir Haji-Akbaria,1

Antifreeze proteins (AFPs) are an interesting class of biomolecules that hinder macroscopic freezing by binding to small ice crystals and blocking their further growth. They were first discovered in the 1960s in arctic fish (1) and have since been observed in a wide range of other organisms, e.g., bacteria (2), fungi (3), plants (4), insects (5), and vertebrates (1, 6), that live in low-temperature environments. AFPs are also of considerable practical interest due to their potential applications in cryopreservation (7), food processing (8), and hydrate inhibition (9). 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. To understand the origin of this complexity, a distinction must be made between traditional antifreeze agents (AFAs), such as salt and ethylene glycol, and AFPs. Traditional AFAs work by reducing Δμ, the thermodynamic driving force for crystallization. This leads to an effective depression in the equilibrium melting temperature, Tf ,eq, proportional to the molal concentration (activity) of the AFA. In contrast, the antifreeze function of an AFP has a purely kinetic origin, emanating from its preferential binding to certain crystallographic planes of the (small) ice crystallites that might exist in the system. This hinders the nucleation and/or growth of such thermodynamically favored ice crystals, and henceforth results in a delay in macroscopic freezing. In this context, the TH of an AFP is defined as the difference between the equilibrium melting temperature and the kinetic freezing temperature of an ice crystal in its presence. This kinetic depression in freezing is typically orders of magnitude larger than the thermodynamic depression emanating from the dissolution of AFP molecules. This makes AFPs such an interesting class of macromolecules

and enables them to operate at concentrations that are orders of magnitude smaller than what is needed for conventional AFAs. By construction, TH is a nonequilibrium quantity that not only depends on AFP concentration but also on other processing factors, such as the cooling rate and the crystallization stimulus (i.e., homogeneous, heterogeneous, or field-induced nucleation) used in a particular experimental procedure. The conventional method of choice for measuring TH is nanoliter cryoscopy (11), which is a two-step process. In the initial preparatory step, a small (∼ 1 nL) volume of the AFP-containing solution is flash-frozen either through rapidly cooling it to −40 °C, or by using a milder quench in conjunction with an external stimulus (e.g., the application of mechanical excitation, or the addition of ice nucleating materials such as silver iodide) (6, 12). The resulting icy mixture is then gradually heated up to a temperature Tm at which the ice crystals start melting. During melting, the system is maintained at Tm until all of the ice melts except for a tiny ice crystal. This is followed by a measurement step in which the system is slowly cooled down to a temperature Tf at which the single ice crystal undergoes a rapid burst of growth. TH is then defined as . At low AFP concentration, ΔT scales linΔT = Tf − Tp mffiffiffiffi early with C, with C the molar concentration of the AFP under consideration. To correct for the concentration dependence of TH, Olijve et al. (10) use the pffiffiffiffi slope of the ΔT vs. C curve as a proxy for TH activity, a quantity denoted by ζ in this commentary. Due to the inherently kinetic nature of TH activity, TH measurements from nanoliter cryoscopy can be sensitive to different features of the preparatory and measurement steps, e.g., the mechanism of ice nucleation (homogeneous, heterogeneous, field-induced), the heating and the cooling rate, and the size and shape of the surviving ice crystal. Furthermore, the samples need to be continuously monitored under the microscope, which makes an automated and quantitatively reproducible determination of TH activity even more difficult. This has led to increased interest in developing alternative assays that would not suffer

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Department of Chemical and Biological Engineering, Princeton University, Princeton, NJ 08544 Author contributions: A.H.-A. wrote the paper. The author declares no conflict of interest. See companion article on page 3740. 1 Email: [email protected].

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A

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Fig. 1. (A) Different crystallographic planes of hexagonal ice. (B) The binding affinities and TH activities of six AFPs studied by Olijve et al. (10). Images of the individual AFPs as well as the ζ values are adapted from ref. 10. Darker boxes correspond to higher values of ζ in any particular assay. TH activities of AFP-III are for a recombinant variant of an ocean pout fish AFP-III, denoted by rQAE in ref. 10.

from such uncertainties. One recently developed alternative is sonocrystallization (13), in which TH activity is estimated in a single measurement, based on the temperature profile of the sample and not the microscopic images of growing or shrinking ice crystals. In sonocrystallization, a few milliliters of the AFP-containing solution are cooled to a temperature of −6 °C. An ultrasonic pulse is then applied to the sample, leading to (cavitation-induced) heterogeneous ice nucleation (14). The latent heat of fusion released upon freezing leads to an increase in Ts, the sample temperature, with respect to Tb, the temperature of the surrounding heat bath. Shortly afterward, Tb is linearly ramped up from −6 °C to +0.4 °C. Due to the latent heat of fusion released as a result of ice growth, however, Ts responds very slowly to changes in Ts − Tb and remains almost constant during the heating process. However, when the AFP-induced kinetic inhibition of freezing kicks in, the ice growth stops, and Ts approaches Tb more quickly. This continues until Ts reaches the equilibrium melting temperature of the solution, at which point a second “melting” plateau is achieved due to the latent heat of fusion adsorbed as a result of melting. The TH is then defined as the difference between these melting and freezing plateaus. Despite the differences between these two assays, one might expect the TH activity estimates obtained from them to correlate strongly with one another. 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 ζcryo and ζsono 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). For instance, DAFP-1 from beetle Dendroides canadensis (15) has a very high cryoscopy TH activity (ζ cryo = 19.8  K · mM−1=2, the second out of the six) and yet a modest sonocrystallization

Haji-Akbari

TH activity (ζsono = 0.32  K · mM−1=2, the fourth out of the six). Contrast this to the recombinant ocean pout fish AFP-III (16) that has a ζ sono = 0.91  K · mM−1=2 (the highest) and yet a ζcryo = 0.90  K · mM−1=2 (the third out of the six, and significantly lower than the first two in the list). 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. 1A, with the binding affinities of the six AFPs considered in ref. 10 specified in Fig. 1B. Such differences in binding affinity are kinetically important, as ice tends to grow at varying rates across its different crystallographic planes. For instance, the rate of ice growth along the a axis (primary prismatic plane) is much faster than along the basal c axis. In nanoliter cryoscopy, cooling rates are slow, and thus both these fast (prismatic) and slow (basal) modes of ice growth are important. Accordingly, an AFP with superior TH activity must be able to bind to both basal and prismatic planes. Indeed, DAFP-1 (15) and MPAFP (M. primoryensis AFP) (2), the AFPs with the two highest ζ cryo values, are both capable of binding to the basal and primary and secondary prismatic planes of ice. Contrast this to the remaining four AFPs that have no known affinity to the basal plane, and therefore have ζ cryo values as little as two orders of magnitude smaller than DAFP1 and MPAFP1. In sonocrystallization, however, freezing occurs very rapidly, and therefore the faster prismatic mode of growth is of higher relevance. Consequently, an AFP’s ability to block the primary prismatic plane is far more relevant in its TH performance than its affinity to the basal plane. Indeed, neither ssAFP1 (17) nor

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wfAFP1 (18) are capable of binding to the primary prismatic plane, and they tend to have the two lowest ζsono 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. Olijve et al. (10) also discuss the importance of both the adsorption kinetics and the surface coverage of AFPs for their TH efficacy. These quantities are far more difficult to determine experimentally in comparison to the binding affinity of an AFP to a particular crystallographic plane, and yet play an equally important role in determining TH activity. For instance, the adsorption kinetics of an AFP to a particular crystallographic plane must be faster than the ice growth rate across that plane in order for such binding to be practically relevant. Further studies are, however, necessary to identify possible connections between these features and the TH activity of proteins and macromolecules with noncolligative antifreeze activity. These findings also pose interesting questions for further research. One interesting possibility is the effect of AFPs on the kinetics and mechanism of ice nucleation. It has been recently suggested that the competition between different polymorphs of ice, and between different crystollagraphic planes of the same polymorph, can play a key role in early stages of nucleation (19). Because different AFPs can bind to different crystallographic planes, it is conceivable for them to affect the nucleation kinetics

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

and mechanism in nontrivial ways. This cannot only affect the texture of the ice crystals that emerge during different TH activity assays but can also add an additional layer of kinetic frustration to the formation of critical ice nuclei in subfreezing environments. To summarize, the findings presented in ref. 10 are of significant theoretical and practical importance, and advance our knowledge on how physicochemical properties of AFPs affect their TH behavior. They also shed light on the possible biological functions of different AFPs in different organisms. For instance, the hyperactive AFPs that bind to both basal and prismatic planes of ice tend to be observed in organisms, such as insects, that can undergo large temperature swings, and must therefore be prepared to withstand fast and slow modes of ice growth. The prismaticbinding AFPs that work best in sonocrystallization are, however, more prevalent in arctic fish, due to their ability to withstand faster modes of ice growth that are relevant in the ice recrystallization inhibition activity necessary under prolonged exposure to subfreezing temperatures in arctic and Antarctic aquatic environments. Finally, these findings underscore the importance of taking ice-binding affinities and the ensuing kinetic implications into consideration when choosing (or designing) an AFP for a particular application. In other words, an AFP that might work very effectively for a given process might not be as effective under alternative processing conditions. It is thus not possible to identify a single metric for rating the antifreeze activity of AFPs, as this quantity such a metric can be very sensitive to the processing conditions under which an AFP is expected to operate.

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Haji-Akbari

Rating antifreeze proteins: Not a breeze.

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