New insights into ice growth and melting modifications by antifreeze proteins

Abstract

Antifreeze proteins (AFPs) evolved in many organisms, allowing them to survive in cold climates by controlling ice crystal growth. The specific interactions of AFPs with ice determine their potential applications in agriculture, food preservation and medicine. AFPs control the shapes of ice crystals in a manner characteristic of the particular AFP type. Moderately active AFPs cause the formation of elongated bipyramidal crystals, often with seemingly defined facets, while hyperactive AFPs produce more varied crystal shapes. These different morphologies are generally considered to be growth shapes. In a series of bright light and fluorescent microscopy observations of ice crystals in solutions containing different AFPs, we show that crystal shaping also occurs during melting. In particular, the characteristic ice shapes observed in solutions of most hyperactive AFPs are formed during melting. We relate these findings to the affinities of the hyperactive AFPs for the basal plane of ice. Our results demonstrate the relation between basal plane affinity and hyperactivity and show a clear difference in the ice-shaping mechanisms of most moderate and hyperactive AFPs. This study provides key aspects associated with the identification of hyperactive AFPs.

Figure 1.

Ice growth patterns at temperatures within the TH gap in solutions of moderately active AFPs, viewed normal to the c-axis. Snapshots were taken during ice growth at temperatures starting from T_m (frame 1) to 0.01–0.1°C below it (frames 2–5). (a) Type I AFP, (b) type II AFP (120 µM), (c) type III AFP (20 µM), (d) AFGPs (500 µM) and (e) IBP from ryegrass (LpIBP) (50 µM). The arrow denotes the direction of the c-axis. The time lapse between images 1 and 5 in (a–e) is 2 min, 3.5 min, 2 min, 40 s and 4 min, respectively.

Figure 2.

Ice growth patterns at temperatures within the TH gap in the presence of moderately active AFPs, viewed along the c-axis. Snapshots were taken during ice growth at temperatures starting from T_m (frame 1) to 0.01–0.08°C below it (frames 2–5). (a) Type I AFP and (b) LpIBP (50 µM). The time lapse between frame 1 and 5 is 35 s and 4 min for (a) and (b), respectively.

Figure 3.

Melting of ice in solutions of moderate AFPs. The melting sequences started from bipyramidal growth shapes at temperatures within the TH gap. The temperature was then raised up to 0.05 °C above T_m. The time lapse between the first and the last frame is 15–50 s. (a) Type I AFP, (b) type II AFP (120 µM), (c) type III AFP (20 µM), (d) AFGPs (500 µM) and (e) LpIBP (50 µM). The arrow denotes the direction of the c-axis.

Figure 4.

Ice melting in solutions of bacterial and insect hypAFPs. The time lapse between the first and the last frame is 15–120 s. The crystals melt in the a-axes direction until the basal planes are very small before melting in the direction of the c-axes is observed. (a–d) The c-axis is denoted by an arrow. (e–h) The c-axis is along the viewer direction. (a,e) GFP-MpAFP (6 µM), (b,f) GFP-sfAFP (2 µM), (c,g) TmAFP (20 µm) and (d,h) GFP-sbwAFP (8 µM).

Figure 5.

Ice growth below the freezing point (burst) in hypAFPs solutions, viewed normal to the c-axis. The temperature of the samples was lowered to the freezing point, and images were taken before the burst (frame 1 counted from the left) and after it (frames 2–4). (a) GFP-MpAFP (6 µM), (b) GFP-sfAFP (2 µM), (c) TmAFP (5 µM) and (d) GFP-sbwAFP (8 µM). (e) The time lapse between frame 1 and 2, 2 and 3, and between frames 3 and 4 is typically 8, 10–120 and 13–57 msec, respectively.

Figure 6.

Tenebrio molitor AFP bind to the basal planes of ice. The fluorescently tagged AFPs visualized both on prism planes and basal plane of ice. The lemon shape was formed as the crystal size became smaller and the flat basal plane slowly melted away (a,b). The arrows show the direction of c-axis. In (c), the c-axis is normal to the view direction. In b,c, the melting was ceased before the basal plane shrank down to a tip.

Figure 7.

Melting versus growth shape comparison. (a,b) Fluorescent images of ice grown in AFP solutions in the presence of Cy5 for background subtraction. A frozen sample was melted until single crystals were formed, and then the temperature was dropped until growth was detected. The arrows designate the crystallographic c-axis. (a) GFP-type III AFP. The bipyramid shapes developed during growth from the eye-shaped crystal. The temperature at which the image was taken is in the TH gap. (b) GFP-TmAFP. The crystal obtained the lemon shape during melting. The growth occurred along the basal direction when the freezing temperature was exceeded. (c) A diagram illustrating the crystallographic directions of the melting shapes of ice in solutions of moderate and hyperactive AFPs.