Physics of Ice Nucleation and Antinucleation: Action of Ice-Binding Proteins

Abstract

Ice-binding proteins are crucial for the adaptation of various organisms to low temperatures. Some of these, called antifreeze proteins, are usually thought to inhibit growth and/or recrystallization of ice crystals. However, prior to these events, ice must somehow appear in the organism, either coming from outside or forming inside it through the nucleation process. Unlike most other works, our paper is focused on ice nucleation and not on the behavior of the already-nucleated ice, its growth, etc. The nucleation kinetics is studied both theoretically and experimentally. In the theoretical section, special attention is paid to surfaces that bind ice stronger than water and thus can be "ice nucleators", potent or relatively weak; but without them, ice cannot be nucleated in any way in calm water at temperatures above -30 °C. For experimental studies, we used: (i) the ice-binding protein mIBP83, which is a previously constructed mutant of a spruce budworm Choristoneura fumiferana antifreeze protein, and (ii) a hyperactive ice-binding antifreeze protein, RmAFP1, from a longhorn beetle Rhagium mordax. We have shown that RmAFP1 (but not mIBP83) definitely decreased the ice nucleation temperature of water in test tubes (where ice originates at much higher temperatures than in bulk water and thus the process is affected by some ice-nucleating surfaces) and, most importantly, that both of the studied ice-binding proteins significantly decreased the ice nucleation temperature that had been significantly raised in the presence of potent ice nucleators (CuO powder and ice-nucleating bacteria Pseudomonas syringae). Additional experiments on human cells have shown that mIBP83 is concentrated in some cell regions of the cooled cells. Thus, the ice-binding protein interacts not only with ice, but also with other sites that act or potentially may act as ice nucleators. Such ice-preventing interaction may be the crucial biological task of ice-binding proteins.

Keywords: antifreeze proteins; freezing; ice nucleation; ice nucleators; ice-binding proteins; ice-binding surfaces; melting; supercooling.

Figure 1

(A) A visualization of interaction of the mIBP83-GFP protein with ice. A comparison of two test tubes with pieces of ice in solutions: with mIBP83-GFP (+IBP) and solely with GFP (−IBP). As seen, mIBP83-GFP is bound to ice, while GFP alone (without mIBP83) is not; see also Figures S2 and S3 in Supplementary Materials as well as in [46]. (B–E) Representative examples of experiments on ice nucleation in different liquid samples in test tubes placed into a thermostat (data for the complete set of experiments are presented in Table 1). The arrows indicate the moment of ice nucleation during cooling. (B) An example of nucleation temperature detection in several cycles of cooling alternating with heating, for 20 mM sodium phosphate buffer, pH 7.0. The ice melting event (seen as the shoulder on the rising part of the curve corresponding to heating) was briefly discussed in [54,55]. But here, we are solely interested in the ice nucleation at cooling—see the beginnings (marked by arrows) of the sharp peaks on the falling parts of the curve. Throughout this experiment, the sample and the test tube remained unchanged, and, as seen, the nucleation temperature was practically the same (±0.4°) for all cycles. Analogous “nucleation peaks” (indicated by arrows) for different samples in different test tubes are shown separately in panels (C–E). (C) Testing an influence of the ice-binding protein on ice nucleation in the buffer. Four blue dashed lines with dashed arrows show cooling of the buffer without mIBP83 (−IBP); four solid red lines with solid arrows show the same buffer supplemented with 0.6 mg/mL mIBP83 (+IBP); this IBP concentration of 0.6 mg/mL is a commonly used antifreeze protein concentration (see, e.g., [56]). The columns of short lines on the left part of the panel indicate the experimental freezing temperatures found in all experiments: blue for the −IBP case, red for the +IBP case, and green for the control protein. The nucleation temperature is seen to be only approximately reproduced after changing the test tube and the liquid sample, but the nucleation temperature range is almost the same for both −IBP and +IBP cases. (D,E) Testing an influence of the ice-binding protein on ice nucleation by potent nucleators. The same experiments with the nucleators CuO and P. syringae, in the same buffer. The ice-binding protein mIBP83 reliably decreased the nucleation temperature. Concentrations/amounts of all substances are given in the caption of Table 1.

Figure 2

Experiments on the effect of an antifreeze protein RmAFP1 on ice nucleation. We have performed 2–5 series of experiments for each liquid (A–D); each series consisted of 10 or more repeats of a cycle of cooling, alternated with heating, without replacing the liquid sample and test tube—as in the experiment demonstrated in panel (B) in Figure 1. The repeat No in each of the series for each liquid is indicated at the bottom of each panel. (A) Water without any proteins, pH 7.0. (B) Water supplemented with 0.04 mg/mL RmAFP1. (C) Water with P. syringae as an ice nucleator (cf. Figure 1E). (D) Water with the nucleator P. syringae supplemented with an antifreeze protein RmAFP1 (0.04 mg/mL). The repeats after No 10 are not shown in the panels for the sake of compactness, but columns of short lines on the left part of the panels indicate the experimental nucleation temperatures found in all repeats of all experiments. The figure shows that, although the antifreeze protein RmAFP1 altered the ice nucleation temperatures in the absence of P. syringae ice nucleator, the impact of RmAFP1 is even more pronounced in the presence of the ice nucleator, because the ± deviations are twice smaller in the latter case.

Figure 3

Localization of the fused protein mIBP83-GFP (+IBP) and GFP alone (−IBP) in SKBR-3 cells. The cells were kept at +37 °C or incubated at +2 °C for 2 h, then fixed and imaged using a laser scanning microscope. The fluorescence images (black background) and the merged “transmittance + fluorescence” images (gray background) are presented for each experiment. The nuclei of some individual cells are marked as nu. The white arrows indicate some of the most pronounced mIBP83-GFP accumulations in some regions of the cooled cells. It is seen that the well-defined accumulation of mIBP83-GFP (and not GFP alone) is only observed at a temperature close to 0 °C, while at +37 °C, both proteins do not accumulate in any small area in the cell.

Figure 4

Schematic drawings of a 3-dimensional (3D) ice nucleus (A), and two kinds (B,C) of 2-dimensional (2D) ice nuclei on underlays of different shapes. The water molecules in ice are shown as light-blue cubes, the surrounding liquid water molecules are shown as light-blue balls, and ice-binding surfaces (underlays) are shown in dark-blue or black. Additional free energies $B_3$ of molecules on different facets of the 3D ice nucleus, in principle, may be somewhat different, since these molecules may have different orientations relative to different facets [39,69]. The 2D nuclei arise on the underlying ice-binding (or ice) surfaces. In extreme cases, the underlays may be smooth (B) or corrugated (C); side views (see insets) show that contacts between the ice molecules inside a layer formed on a smooth underlay are strong, while contacts between the ice molecules inside a layer formed on a corrugated underlay are weak, while the contact of this ice layer with the underlay is stronger in case (C) than in case (B). Respectively, the additional free energy of a border molecule of the layer arising on a smooth underlay $\left(B_2^{\prime }\right)$ is high, while the additional free energy of a border molecule of the layer arising on a corrugated underlay $\left(B_2^{″}\right)$ is low. Thus, ice nucleation time drastically decreases on corrugated surfaces as compared to smooth ones.