Effect of annealing time of an ice crystal on the activity of type III antifreeze protein

Abstract

Antifreeze proteins (AFPs) are a structurally diverse class of macromolecules that interact with water molecules located in the surface of an ice crystal at temperatures below the melting point of the solution [1]. Interaction of a substantial number of AFPs with the ice surface modifies the shape of the ice crystal, resulting in unique morphologies, such as a hexagonal bipyramid or hexagonal trapezohedron [2]. AFPs inhibit ice growth by adsorbing onto the ice surface (adsorption-inhibition model) [3,4] as the temperature is lowered, and such inhibition becomes insufficient below a certain temperature that is favourable for the initiation of crystal growth. For AFP solutions, this 'ice-growth initiation temperature' (T ini ) is different from the melting point (T m ) of the ice crystal, and the T ini is referred to as the nonequilibrium freezing point (nonequilibrium T f ). The difference between T m and the nonequilibrium T f is defined as thermal hysteresis (TH), and is generally used as a measure of the growth inhibition ability of an AFP [5]. Therefore, determination of the T ini and T m of a seed ice crystal is an essential procedure to evaluate the TH activity of AFP. However, the mechanism of ice binding that alters the TH value remains unclear [6].The TH value of AFPs has been evaluated using a nanolitre osmometer and the 'capillary technique' [5,7]. In the former, submicrolitre volumes of an AFP solution are introduced into an oil droplet, the temperature of which is controlled by a Peltier device. The TH value is determined by observing the growth of an Antifreeze proteins (AFPs) possess a unique ability to bind to a seed ice crystal to inhibit its growth. The strength of this binding has been evaluated by thermal hysteresis (TH). In this study, we examined the dependence of TH on experimental parameters, including cooling rate, annealing time, annealing temperature and the size of the seed ice crystal for an isoform of type III AFP from notched-fin eelpout (nfeAFP8). TH of nfeAFP8 dramatically decreased when using a fast cooling rate (0.20°CAEmin )1 ). It also decreased with increasing seed crystal size under a slow cooling rate (0.01°CAEmin), but such dependence was not detected under the fast cooling rate. TH was enhanced 1.4-and 2.5-fold when ice crystals were annealed for 3 h at 0.05 and 0.25°C below T m , respectively. After annealing for 2 h at 0.25°C below T m , TH activity showed marked dependence on the size of ice crystals. These results suggest that annealing of an ice crystal for 2-3 h significantly increased the TH value of type III AFP. Based on a proposed adsorption-inhibition model, we assume that type III AFP undergoes additional ice binding to the convex ice front over a 2-3 h time scale, which results in the TH dependence on the annealing time.Abbreviations AFGP, antifreeze glycoprotein; AFP, antifreeze protein; nfeAFP8, an isoform of type III AFP from Notched-fin eelpout; TH, thermal hysteresis.

Crystal Structure of an Insect Antifreeze Protein and Its Implications for Ice Binding

“…The activity measured in vitro is likely to be a significant underestimation of the natural activity because the cooling rate and the initial ice crystal size are greater than those occurring in nature. It has been demonstrated that these two parameters are inversely proportional to the thermal hysteresis activity measured by experiment (48). Importantly, whereas other hyperactive antifreeze proteins have proven difficult to produce in large quantities, RiAFP is expressed in E. coli at concentrations of up to 50 mg/liter of cell culture (24).…”

Microfluidic experiments reveal that antifreeze proteins bound to ice crystals suffice to prevent their growth

“…The existence of a slow adsorption and desorption rates was suggested recently by Kubota to explain the time-dependent behavior of the FH activity (34). The dynamic nature of the AFP adsorption was previously reported by other groups for two moderately active AFPs (35,36). Thus, to ensure that the ice surface is well protected by AFPs, we exposed ice crystals to high concentrations of GFPTmAFP for at least 10 min before removing AFPs from the solution.…”

Crystal Structure and Mutational Analysis of Ca2+-Independent Type II Antifreeze Protein from Longsnout Poacher, Brachyopsis rostratus

“…66 Seven microliters of AFP dissolved in 20 mM Tris-HCl with 4 mM EDTA (pH 8.0) was placed in a glass capillary tube with an inner diameter of 0.92 mm (outer diameter of 1.39 mm) and length of approximately 6 mm. After each end of the tube was sealed with mineral oil, the capillary tube was placed in a copper capillary holder.…”