Adsorption and diffusion of Antifreeze Glycoprotein molecules at ice/water interface

Antifreeze Proteins (AFPs) and Glycoproteins (AFGPs) are found in nature in many cold weather organisms including fish, amphibians, plants and insects. These proteins suppress the freezing temperature of the blood serum in fish just enough to keep them from freezing in their supercooled environments while the melting temperature remains unchanged (hysteresis) and inhibit ice recrystallization enough to reduce damage in freeze tolerant organisms, making them essential for survival. To date workers have described at least five distinct classes of these proteins, AFP types 1 to 4 and AFGPs, as well as two distinct types of insect AFPs. The Type 1 and Type 2 AFPs are alanine and cysteine rich, respectively, while the AFGPs are alanine rich as well as glycosolated. They exist in a variety of structures ranging from alpha- or beta-helical to globular and yet to some extent they all accomplish the same function. These proteins are thought to bind to the surface of ice and inhibit growth. Although, this mechanism has recently been challenged by mutation experiments, where the residues thought to bind to the ice lattice were replaced with hydrophobic ones and these retained nearly all of the antifreeze activity. However, there is a consensus that this is a surface phenomenon, but no clear evidence for the exact nature of the mechanism is available and is necessary to understand how these proteins work. In this work, we use 3-d confocal microscopy to gain insight into the antifreeze interaction with the ice/solution interface. Single ice crystals are grown in solution from a capillary in the presence of antifreeze proteins labeled with Fluorescein Isothiocyanate (FITC). At 5 micro-g/ml quantities of AFGP we see a clear adsorption at the prismatic planes with growth stopped while the temperature is in the hysteresis region. When we lower the temperature below the hysteresis region growth continues while the protein is rejected from the crystal. At higher concentrations the gross morphology can vary quite dramatically, but the proteins are still rejected from the solid. This contradicts previous understandings that the mechanism for antifreeze action must be a tight irreversible binding. Furthermore, the conformational change of Antifreeze Glycoprotein (AFGP) molecules during ice formation on the AFGP thin film was followed by Attenuated Total Reflection (ATR)-FTIR (Fourier Transform Infrared) spectroscopy. The ATR-FTIR difference spectrum of the frozen and supercooled samples showed that helical conformation of AFGP molecules is predominant at the ice-water interface.
資料番号: AA0063349002