Figure 1.
Ice crystal shaping during growth in the presence of (A) 100 mM sodium acetate (pH 4.2), (B, D, F) ZRAH (30 g/L) and (C, E, G) ZRA (13.3 g/L) solutions.
Figure 2.
The effect of ZRA and ZRAH on the growth rate of ice crystals along the a-axis.
The X-axis represents the temperature below Tm. The Y-axis represents the rate of growth of the radius of the ice crystal. Each point represents the average of at least 3 measurements. (B) A magnified view of the area enclosed by the black rectangle in A. The horizontal dash-dot line indicates a growth rate of 0.02 µm/sec, which is the threshold value used to define the TH gap. The arrows indicate the semi-TH activity according to this modified definition. Note that according to the old threshold definition (0.2 µm/sec) the TH activity in the a-axis direction would be >0. 1°C.
Figure 3.
Comparison of the effects of ZRAH and ZRA on the ice growth rates along the c-axis.
The X-axis represents the temperature below Tm. The Y-axis represents the rate of growth of the radius of a seed ice crystal 20 µm in length. For ZRAH solutions, growth at ΔT >0.1°C could not be measured due to crystal orientation.
Figure 4.
Ice recrystallization inhibition by ZRA and ZRAH.
LC-PolScope images for (A) ZRA at pH 4.2, (B) ZRAH at pH 4.2, (C) 100 mM sodium acetate pH 4.2 (negative control), and (D) AFP III (40 µM solution). The colors represent the optical axis orientations of the ice which was determined by the Abrio birefringence detection system. The color map in the bottom right corner corresponds to the respective birefringence orientation. The images in the first and second columns show the initial frozen state at 4× and at 50× magnifications, respectively. Images in the last three columns were collected at an annealing temperature of –3°C, at times 0, 10, and 60 minutes. Note the grain boundaries that appeared during annealing only in the buffer sample. These boundaries shifts indicate recrystallization process.
Figure 5.
Proposed mechanism for ice recognition by ZRA and ZRAH.
A–B. Representation of the Structure of AFP from mealworm beetle (Tenebrio molitor) (TmAFP), showing the backbone as a ribbon and the threonine residues forming the ice-binding face in a ball-and-stick model. Oxygen atoms are presented in red and carbon atoms in gray. (A) Side view of the protein. (B) Cross-section of the structure. (C–D) Models suggested for the interactions of ZRA and ZRAH with ice. See text for details.