Contrasting Behavior of Antifreeze Proteins: Ice Growth Inhibitors and

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Chemical and Dynamical Processes in Solution; Polymers, Glasses, and Soft Matter

Contrasting Behavior of Antifreeze Proteins: Ice Growth Inhibitors and Ice Nucleation Promotors Lukas Eickhoff, Katharina Dreischmeier, Assaf Zipori, Vera Sirotinskaya, Chen Adar, Naama Reicher, Ido Braslavsky, Yinon Rudich, and Thomas Koop J. Phys. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.jpclett.8b03719 • Publication Date (Web): 11 Feb 2019 Downloaded from http://pubs.acs.org on February 12, 2019

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The Journal of Physical Chemistry Letters

Contrasting Behavior of Antifreeze Proteins: Ice Growth Inhibitors and Ice Nucleation Promotors Lukas Eickhoff,†# Katharina Dreischmeier,†# Assaf Zipori,‡# Vera Sirotinskaya,§ Chen Adar,§ Naama Reicher,‡ Ido Braslavsky,§ Yinon Rudich,‡ Thomas Koop†* † Bielefeld University, Faculty of Chemistry, Bielefeld, Germany. ‡ The Weizmann Institute of Science, Department of Earth and Planetary Sciences, Rehovot, Israel. § The Hebrew University of Jerusalem, Robert H. Smith Faculty of Agriculture, Food and Environment, Institute of Biochemistry, Food Science and Nutrition, Rehovot, Israel. # These authors contributed equally to the work.

AUTHOR INFORMATION *Corresponding Author: [email protected]

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ABSTRACT: Several types of natural molecules interact specifically with ice crystals. Small antifreeze proteins (AFP) adsorb to particular facets of ice crystals thus inhibiting their growth, while larger ice-nucleating proteins 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 antifreeze proteins and ice-nucleating proteins is their molecular size, which is a very good predictor of their ice nucleation temperature.

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The freezing of ice from water and aqueous solutions is among the most prevalent liquid-tosolid phase transitions on Earth, with beneficial effects such as the initiation of precipitation in the atmosphere, as well as adverse effects such as frost damage in living organisms. In response, nature has developed molecules that directly interfere with the formation and growth of ice crystals. Freezing is a two-step process consisting of an initial ice nucleation event, i.e. the formation of a first stable ice embryo, and the subsequent growth of this ice embryo into a macroscopic crystal. Natural ice-nucleating molecules – most often proteins – can act as catalysts for heterogeneous ice nucleation by providing an active site that stabilizes and, thus, facilitates the formation of stable ice embryos.1–8 In contrast, nature also developed antifreeze proteins that can adsorb to preexisting or newly-formed ice crystals, thereby inhibiting or even entirely stopping their growth.9–11 The origin of the apparently contrasting behaviors of antifreeze proteins (AFP) and ice-nucleating proteins (INP) are hotly debated, in particular since the interaction of their active sites with ice crystal facets appear to be very similar.9,11–13 Despite the fact that there are no experimental but only modeled crystalline structures for ice-nucleating proteins,14 the high similarity of the active site structure of an hyperactive insect AFP from Tenebrior molitor (TmAFP) and that of a bacterial ice-nucleating protein (INP) from P. syringae spurred the discussion of whether INPs may simply be larger versions of AFPs.11,13–16 Indeed, a similar interaction with water and ice of the active site of the two groups of proteins has been supported by spectroscopic experiments and by recent molecular dynamics simulations.12,17,18 There is indeed limited experimental support for this notion, indicating that larger AFP can show ice nucleating properties. For example, partially purified mixtures containing a 164 kDa AFP showed ice nucleation activity, but a significantly shortened fragment (72 kDa) lost its ability to nucleate ice.6 A shortened polypeptide fragment from an INP from P. syringae also showed



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antifreeze properties.7,19 Likewise, clusters and aggregates of INPs improved the ice-nucleating activity, i.e. the larger aggregates had a higher freezing ability.4,8,20 In contrast, macroscopic silicon or aluminum substrates decorated with an AFP monolayer enhanced the ice nucleation temperature by a few degrees when compared to decoration with the non-active side of the protein, or with the bare substrate, indicating that macroscopic AFP-occupied surfaces can trigger ice nucleation.21,22 However, here we focus on the behavior of proteins dissolved in a solution. In the following we show that two different types of widely studied antifreeze proteins, one from an Arctic fish (type III AFP from ocean pout) and one from a beetle (TmAFP from T. molitor) exhibit both functions in aqueous solutions: they inhibit ice growth as shown previously,9,10,23–26 but they also exhibit the ability to nucleate ice in solutions. There have been several experimental studies on the effects of dissolved AFPs on ice nucleation, most of them addressed the question of whether AFPs can reduce or entirely inhibit the activity of other ice nucleators,27–30 but to the best of our knowledge none of them presented unambiguous experimental proof for ice nucleation triggered by the dissolved AFPs themselves. The latter experiments either employed inverse (water-in-oil) micro-emulsions with picoliter droplets and, thus, the effect on the ice nucleation temperature was too small to be significantly different from the homogeneous freezing signal;27,29,30 or they employed microliter droplets, in which heterogeneous ice nucleation triggered by supporting surfaces or particulate impurities are inevitable and, hence, mask any anticipated weak effect of AFP or synthetic mimics.28,31–33 There is one study devoted to the mobility of water molecules in the hydration layer of dissolved TmAFP using magnetic relaxation dispersion, which also discusses freezing as a side aspect.34 It is stated that in emulsified pure water droplets, the water resonance peak strongly reduced at about -35.5°C. This was taken as an evidence for homogeneous freezing; in the emulsified TmAFP solution, a strong peak reduction was observed


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~5 °C higher than for water, while other proteins showed a more moderate effect (~2 °C). The authors thus suggested that the larger effect of the TmAFP solution may be attributed to the ice affinity of TmAFP and that TmAFP may nucleate ice at these low temperatures. Below, we provide unambiguous experimental proof for these speculations. We used a newly developed microfluidic device that employs nanoliter droplets,35 i.e. droplets that are small enough such that most of them are devoid of impurities and hence freeze at the homogeneous ice nucleation temperature, but simultaneously are large enough to contain sufficient amounts of AFP molecules to enhance the probability of triggering heterogeneous ice nucleation. In Figure 1 we show the results of ice recrystallization inhibition efficacy studies of the AFP using the IRRINA (Ice Recrystallization Rate INhibition Analysis) assay.23,36 Briefly, a thin polycrystalline ice film is created and then tempered at -8 °C for two hours. During that time, the Ostwald ripening process of larger ice crystals that grow at the expense of smaller ones is observed by measuring the average ice crystal size as a function of time, from which the ice recrystallization rate constant kl0 is obtained and plotted in Figure 1 as a function of AFP concentration. While at low AFP concentration kl0 is not affected, at higher concentrations the AFP first inhibit, and then entirely stop the ice recrystallization process as evident from the fact that kl0 drops to zero.



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Figure 1. Ice recrystallization rate constants obtained with IRRINA as a function of AFP concentration for type III AFP (magenta circles, original data from Ref.23) and TmAFP (green squares). The lines are Hill-type fits to the data, resulting in efficacy concentrations (dashed lines originating from the inflection points of the fits) of ci = 0.5 µmol L-1 for type III AFP and ci = 1.6 µmol L-1 for TmAFP. Note that the difference in kl0 for zero concentration stems from slightly different protocols for the previous measurements23 of type III AFP and the current ones on TmAFP; it is, however, not relevant for the determination of ci.

The inflection point of the Hill-type fit to the data indicates the concentration ci that represents the ice recrystallization inhibition efficacy of a particular AFP. Both type III AFP (ci = 0.5 ±0.2 µmol L-1) as well as TmAFP (ci = 1.6 ±0.7 µmol L-1) are similarly effective as other AFP such as type I AFP, but less effective than antifreeze glycoproteins AFGP (AFGP1-5 in particular).23 We also investigated MBP-TmAFP, a version of TmAFP that contains a large maltose binding protein domain, and found that it was slightly more effective (ci = 0.5 ±0.2 µmol L-1), see Figure S1 in the Supporting Information.


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Thereafter, we investigated whether type III AFP and TmAFP affect ice nucleation in aqueous solutions. According to previous suggestions, the temperature of heterogeneous ice nucleation triggered by an ice-nucleating molecule increases with its molecular weight.4,5 Therefore, we expected the relatively small AFP with molecular weights of 8.1 kDa (type III AFP) and 8.4 kDa (TmAFP) to show, if at all, only a minor enhancement in the ice nucleation temperature, i.e. only marginally above the temperature of homogeneous ice nucleation, which is about -36 °C for nanoliter-sized droplets at moderate cooling rates. Hence, for our study, a technique is required in which the majority of droplets indeed approach the homogeneous ice nucleation temperature. While this usually cannot be achieved with microliter volume samples, it can be established with nanoliter volume samples in microfluidic devices,35,37–41 see a detailed discussion in the Supporting Information and Figure S2. For the purpose of the current experiments we employed the WISDOM (WeIzmann Supercooled Droplets Observation on a Microarray) device developed recently,35 see experimental methods. Briefly, monodisperse aqueous droplets with a diameter of about 90 µm (~nanoliter volume) were produced within a microfluidic device by pumping the aqueous phase through a central channel and an organic phase containing a non-ionic surfactant through two perpendicular channels in a 4-way junction (see Fig.1a in Reicher et al.35). The droplets were fed into an array of microscopic chambers, that each host one nanoliter droplet, see Figure 2a left image. Subsequently, the entire microfluidic device with the droplet array is placed onto a cold stage of an optical microscope in bright-field transmission mode, so that upon cooling, ice nucleation of individual droplets can be detected by a change in droplet opacity upon freezing, see Figure 2a middle and right image. With this setup, the majority of pure water droplets nucleate ice close to the homogeneous ice nucleation



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temperature predicted by a recent semi-empirical model employing classical nucleation theory42 (Figure S2 in the Supporting Information).

Figure 2. Ice nucleation in AFP solution droplets. (a) Microphotographs of droplets containing 0.08 mg mL-1 of TmAFP at various temperatures during cooling. Frozen droplets appear dark in bright-field transmission of the microscope. (b) Frozen fraction of droplets containing different concentrations of TmAFP (green) in PBS buffer (blue). (c) Frozen fraction of droplets containing


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type III AFP at 66 µmol L-1 (magenta) in PBS buffer (orange). Points represent average values and their standard deviation of four individual experiments with type III AFP and three with pure buffer (dashed lines), see also Figure S3 in Supporting Information.

In Figure 2b and 2c we show the frozen fraction of droplets as a function of temperature, i.e. the cumulative fraction of droplets frozen upon cooling at a particular temperature. Because the AFP were dissolved in a buffer rather than in pure water, we also investigated the ice nucleation in droplets of the two employed buffers, see Figures 2b and 2c. As predicted from theory,43,44 the buffer solutions froze at temperatures lower than pure water due to a non-ideal colligative effect, see Figure S2 in the Supporting Information. Most interestingly, however, upon addition of AFP to the buffer solutions we observed a clear increase in ice nucleation temperature. At TmAFP concentrations between 0.5 and 95 µmol L-1 (corresponding to 0.004 and 0.8 mg mL-1, respectively) 50% of the droplets had crystallized upon cooling to between -33.9 °C and -37.1 °C compared to the buffer’s 50% freezing temperature of -38.4 °C, see Figure 2b. A clear concentration dependence is observed, i.e. higher TmAFP concentrations lead to higher freezing temperatures. Note that the increase in the 50% freezing temperature observed here for the highest TmAFP concentration is similar in magnitude to that reported in the Modig et al.34 study discussed above, and for the lower concentration the observed increase is in agreement with a value of 2°±1°C suggested in recent modelling calculations.45 Whether or not this concentration dependence is due to clustering of TmAFP to dimers, trimers etc. at higher concentrations cannot be answered at present, but we note that according to the modelling calculations by Qiu et al.45, the observed increase at high concentration would be consistent with such dimer formation.



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Similarly, also type III AFP at 66 µmol L-1 (0.53 mg mL-1) leads to ice nucleation temperatures significantly higher than that of the buffer solution, see Figure 2c, although the scatter between individual experimental runs is larger and only three of the four individual runs exhibit ice nucleation temperatures that are higher than each of the three individual buffer runs up to a frozen fraction of 1. (All four type III AFP runs freeze at higher temperature than the entire curves of two of the three buffer runs, and they are all higher than the third buffer run up to a frozen faction of ~0.8, see Supplementary Figure S3). On average, for the type III AFP solutions, 50% of the droplets had crystallized upon cooling to between -35.0 °C and -37.4 °C, while the buffer froze at -37.8 °C. Heating one of the type III AFP solutions for 30 min at 90 °C reduced its ice nucleation activity significantly and was consistent with a concomitant reduction in the thermal hysteresis activity (see Figure S4 and corresponding text in the Supporting Information), supporting the fact that the observed ice nucleation was indeed triggered by the AFP. We note that the shape of the ice nucleation curve of type III AFP is different from that of TmAFP. We surmise that the steep increase in frozen fraction at lower temperature is due to ice nucleation induced by individual AFP, while we cannot exclude the possibility that the more shallow increase below about -30 °C is due to ice nucleation triggered by smaller oligomeric clusters of the AFP. Indeed, in a recent detailed modelling study of ice nucleation by AFP and INP it was shown that even dimers and trimers – when arranged in favorable geometry – can increase the ice nucleation temperature by several degrees,45 just as observed in Figure 2c. Such small clusters may actually form only upon cooling, and if they are indeed present, the observed frozen fraction curve suggests that they occurred in less than half of the droplets studied. We note that such small clusters cannot be eliminated by filtration.


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To the best of our knowledge, the measurements shown in Figure 2 present the first unambiguous and detailed experimental evidence that naturally-occurring AFP smaller than 10 kDa can also act as ice nucleators in aqueous solutions, in agreement with a previous study of a much larger 164 kDa AFP.6 These experiments thus corroborate the long-standing suggestion that the principal difference between AFPs and INPs is their size, which in turn leads to rather different functional behaviors in their natural habitat. We note that the interaction of both proteins types with water and ice may actually be quite similar. It is likely steered by the same type of recognition and interaction process between molecular moieties of the AFP or INP on the one hand, and the ice crystal lattice and its particular facets on the other.12 To further support the notion of similar interaction of ice-active molecules with ice we show in Figure 3 a plot that depicts a strong correlation between the size of various AFP and INP with the size of an ice embryo cap formed on the protein surface as a function of temperature determined from classical nucleation theory (CNT). For that purpose, we first use CNT to calculate the size of an ice cap on a flat surface (see schematic in Figure 3a), which is similar to the arrangement used in more detailed heterogeneous ice nucleation modelling on INP.45 The radius of the circle of contact with the surface, , can be calculated using geometric arguments

⋅ sin from the

contact angle between ice and the surface, , and the radius of the critical ice embryo

from a

previous CNT model46 (see Figure S5 and related parameterization in Eq. (S1) in the Supporting Information). We note that in CNT the radius of the critical ice embryo is identical for homogeneous and heterogeneous nucleation. However, the barrier is reduced for the heterogeneous case.47,48 We further note that for all contact angles smaller than 90°, smaller than

. The temperature dependence of the corresponding diameter

2

will be is shown

as the red lines in Figure 3c for four different contact angles. For example, at a temperature of -



18.9 °C

is about 3.0 nm, and for a contact angle of

45° the corresponding value of

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is

about 2.1 nm. Next, we approximate the protein by a cube with an edge length . One of the surface squares of the cube is the active site, see schematics in Figure 3b. In this geometry, the ice cap perfectly fits onto the square when the circle diameter

2 is equal to the square edge . We

can calculate from the molecular weight of a protein, see Eq. (S2) in Supporting Information, and then plot these values for literature data3,4,6–8 of various ice-nucleating molecules of a particular molecular weight at their ice nucleation temperature (blue symbols in Figure 3c). Some of the data are similar to those shown in Pummer et al.5, but we also added very recent data of an engineered synthetic ice-nucleating protein fragment INpro from P. Syringae7 at -24 and -26 °C.

Figure 3. Heterogeneous ice nucleation on molecular ice nucleators. (a) Geometry of an ice cap formed on a flat surface and its relation to the critical embryo radius. (b) Geometry of an ice cap (red) forming on a flat surface of a simplified cubic protein (blue). The matching diameter of the ice cap contact circle

and the size of the protein

are indicated. (c) The ice nucleation

temperature of molecules of various size (blue data) strongly correlates with the diameter of the


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critical ice cap (red lines) for different contact angles , as calculated from CNT.46 The new data for type III AFP and TmAFP are shown in the lower left corner.

Moreover, we also added the new ice nucleation data of type III AFP and TmAFP presented above, see the darker blue symbols in the lower left of Figure 3c. These new AFP data are the points farthest on the low-temperature side of the plot, in line with the fact that the investigated AFP have the smallest molecular weight in that comparison. Thus, together with the INpro data, our new AFP data extends the previously available temperature range for proteins in that plot by about 20 °C, i.e., by a factor of 2. All data combined strongly indicate that the size of the icenucleating molecules and their ice nucleation temperature are well correlated to the critical ice cap size for a contact angle of

45°

15°. The new AFP data points follow the red lines,

suggesting that they behave similarly to the larger ice-nucleating molecules and clusters. Overall, the good agreement of the data points with the correlation lines suggests that it is indeed primarily the size of an ice-active molecule that determines the temperature at which it triggers ice nucleation, irrespective of whether in its natural environment it functions as an AFP or as an INP. Moreover, the similarity of

of the critical ice cap and

of the ice-nucleating molecule as a

function of temperature may imply that the size of the active site responsible for triggering ice nucleation is likely similar to that of its host molecule, e.g. the protein, as well as that of the ice cap. This correspondence may also suggest that a protein expressed by an organism in nature is apparently just about large enough to provide an appropriately sized active site for its interaction with ice.



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Evidently, there is some amount of scatter in the data around the correlation lines, which may be attributed to several causes: variation in the ice nucleation temperature with droplet size, INP concentration or cooling rate; uncertainty in the molecular weight of the ice-active molecule and the approximation of its size ; uncertainty in the calculation of the critical ice embryo radius and, thus, of

from CNT; finally, the molecular weight may be larger for molecules that contain a

significant fraction of moieties that are not involved in the ice-active site. Considering these potential limitations, the observed clear relationship shown in Figure 3c is indeed striking. It is intriguing that size appears to be the primary predictor for the ice nucleation temperature, and not the strength of the molecule/ice embryo interaction, i.e. the Gibbs free energy difference between a bare ice embryo and an ice embryo cap stabilized by an ice-nucleating molecule. Part of the scatter in the data may also be due to differences in this Gibbs free energy of stabilization ∆ of the ice cap by different molecules, as represented by lines of different contact angle, although the very good correlation between the blue data points and the red lines suggests that this appears to be a secondary effect. We note that the data for ice-nucleating polysaccharides at -18 °C is somewhat higher, suggesting that, compared to a protein, a polysaccharide is less structured thus revealing a smaller compatibility and larger contact angle. Moreover, the larger bacterial icenucleating proteins at higher temperatures seem to better fit the line for a contact angle of 30°. Whether this indeed indicates a better compatibility with the ice cap or is due to uncertainties as discussed above is not clear at present. In order to prove experimentally the relatively weak overall dependence of variation in ∆

, more detailed studies are required on aqueous solutions of

clearly defined and characterized monodisperse molecules in order to distinguish experimental variations from those of the actual molecule/ice interaction strength. Hence, biochemical means for producing larger AFP molecules or smaller INP molecules may be one avenue to proceed.


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In conclusion, our data clearly show that antifreeze proteins exhibit contrasting behavior: they inhibit the growth of preexisting ice crystals just as expected, but they can also promote the nucleation of new ice crystals from supercooled solutions. Overall, our study – and in particular the analysis of Figure 3c – provides a roadmap for the development and synthesis of molecules with an ice nucleation temperature customized for a particular application.



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EXPERIMENTAL METHODS Protein production. Expression of recombinant MBP-TmAFP (MBP-His6-TEV-TmTHP fusion described in Bar et al.49) in Origami-B E. coli (DE3) plysS strain (Novagen) and subsequent workup and purification were done as described previously.49,50 MBP-TmAFP plasmid was generously donated by Deborah Fass (The Weizmann Institute of Science, Rehovot, Israel). TmAFP was produced by cleaving purified MBP-TmAFP via digestion with TEV protease (ProTEV Plus, Promega, USA) overnight and removing the MBP with His6 by binding to Ni-NTA resin (Novagen). The final solution of MBP-TmAFP (1.6 mg ml-1) was prepared in 1xPBS buffer (pH 7.4), that of TmAFP (0.8 mg ml-1) in 2.7(±0.3)xPBS buffer. Expression of recombinant type III AFPwas based on gene PBD: 1HG7_A with an addition of EHHHHHH (6xHis tag) donated by Peter L. Davies, Queens University. Theplasmid was performed as described for MBP-TmAFP, with the difference that the plasmid containing the recombinant type III AFP was transformed into BL21-DE3-PlysS E. coli strain grown in a stirred 1 L tank fermenter. The obtained supernatant was purified by two rounds of falling water ice affinity purification as described previously50 or by nickel affinity chromatography, in which type III AFP was bound to Ni-NTA resin, washed with lysis buffer, eluted in one step with a buffer containing 200 mM imidazole, and finally dialyzed against PBS to yield a solution of 0.53 mg ml-1 type III AFP in 1xPBS buffer. For the IRRINA measurements, type III AFP from natural sources (molecular weight: 6.5 kDa) were obtained commercially from A/F Protein (Waltham, MA). Note that such natural samples will be a mixture of several different isoforms. For details see the Supporting Information. Ice recrystallization measurements. The recrystallization inhibition efficacy of TmAFP and MBP-TmAFP was evaluated in 45 wt% aqueous sucrose solutions using the IRRINA assay,23,36 the original data for type III AFP were obtained previously.23 In IRRINA a thin sample film with 1.5


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µl of the sample solution was pressed between two glass coverslips and was then transferred into a temperature-controlled cryostage linked to an optical microscope equipped with a digital camera. The sample was frozen by quickly cooling to -50 °C and annealed at -8 °C for 120 min during which time the cubic mean radius of all ice crystals in the field of view was analysed as a function of time. Ice nucleation measurements. These measurements were performed with WISDOM microfluidic devices.35 The various AFP solutions were introduced into the device producing aqueous droplets about 90 µm in diameter suspended in a 2 wt% solution of an emulsifier (Span 80) in mineral oil. The device was then cooled at a rate of 1 °C min-1 on a cryostage under a microscope and the freezing temperature of each individual droplet was recorded with a digital camera and determined from subsequent image analysis.

ASSOCIATED CONTENT Supporting Information. More details on sample preparation, experimental methods and calibration, additional experiments and a parameterization of the critical ice embryo size and protein size. AUTHOR INFORMATION Notes: The authors declare no competing financial interests. Author Contributions: The study was conceived by I.B., Y.R. and T.K. with contributions from all authors. L.E. performed the TmAFP ice nucleation experiments, and established and calibrated the WISDOM device at Bielefeld University. K.D. performed the IRRINA experiments. A.Z.



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performed the type III AFP ice nucleation experiments, and with support from N.R. established and calibrated the WISDOM device at the Weizmann Institute. V.S. prepared the TmAFP and MBP-TmAFP samples and C.A. prepared the type III AFP samples. N.R. prepared the WISDOM chips. All authors contributed to the analysis and discussion of experimental data and their interpretation. T.K. wrote the article with input from all authors.

ACKNOWLEDGMENT The authors thank C. Budke for provision of original IRI data for type III AFP, S. Ben Bassat for TH measurements of type III AFP, H.P. Dette for helpful comments, and the anonymous referees for helpful comments and for pointing us to the statements on ice nucleation in the paper by Modig et al. The authors are also grateful for funding by the German Research Foundation (DFG) through the research unit FOR 1525 (INUIT) for T.K. (KO 2944/2-2) and a DFG Mercator Fellowship for Y.R.; Y.R. also acknowledges support from the Estate of David Levinson, and I.B. support from the Israel Science Foundation (ISF grant No. 930/16).


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