Cool molecules: unraveling the secret of anti-freeze proteins

Anti-freeze proteins (AFPs) and anti-freeze glycoproteins (AFGPs) have a surprisingly strong effect on the freezing point of water. Even very low concentrations are sufficient to lower the freezing temperature by 2 degrees Celsius. To reach the same freezing point depression using NaCl would require a concentration of ~0.5 M. AFPs and AFGPs have evolved in fish, insects, plants, fungi, bacteria, and enable these organisms to survive in polar and near-polar areas. Nowadays these proteins also find applications in food industry, for instance to prevent recrystallization in ice cream. The lowering of the freezing point of water by adding solutes is a quite general phenomenon. For practically all solutes (salts, sugars) this effect results from the entropy of mixing and does not depend on the nature of the solute (colligative effect). However, for AFPs and AFGPs the effect on the freezing point is much larger (up to 500 times) than the colligative effect and shows a strong hysteresis, meaning that only the freezing point and not the melting point is being depressed. Therefore the depression of the freezing point due to AFPs and AFGPs is thought to be the result of highly specific interactions between these glycoproteins and the growing ice crystals. As such, the freezing point depression by proteins constitutes a highly non-colligative effect. Protein-water interactions are of general interest owing to the importance of protein hydration for protein function; AFPs and AFGPs form an extraordinary example of this coupling that is sufficiently strong and specific that the protein controls macroscopic thermodynamic properties of water by steering the water structure and dynamics. Despite their apparent importance and industrial relevance, the mechanisms by which AFPs and AFGPs depress the freezing point have remained poorly understood. Although substantial information presently exists on static protein structures and thermodynamic properties of these systems, molecular scale information on the dynamics of the conformations of the (glyco)proteins and their hydration shells is extremely scarce. Here we propose to study the molecular mechanisms by which AFPs and AFGPs lower the freezing temperature with advanced (nonlinear) spectroscopic techniques like 2D polarization-resolved vibrational spectroscopy, surface sum-frequency generation and transient THz spectroscopy. These techniques enable the label-free study of the structural dynamics of the (glyco)protein conformations, the ice surfaces and the hydration shell. The power of these techniques in studying molecular-scale dynamics of aqueous systems has been demonstrated in many studies, including work by our group. We will use these techniques to study several key elements in the functioning of AFPs and AFGPs. One of these elements is the change in conformation of the AFPs and AFGPs when they adsorb to ice. The conformational dynamics of the (glyco)proteins will be accompanied by changes in the structure and dynamics of their hydration shells that may be essential in the process of adsorption. Therefore another important element to be studied is the structure and dynamics of the hydration shell. These studies may for instance reveal whether some parts of the hydration shell already acquire ice-like properties in the liquid phase before the AFP/AFGP adsorbs. The AFP/AFGP and its hydration shell will adsorb to ice and may alter the structure of the ice surface. Therefore we will also study the structure of the top layers of the ice under adsorbed AFPs and AFGPs. Finally, as AFPs and AFGPs function in the presence of dissolved salts and sugars, we will study how these other solutes influences their functioning. The results of the experimental studies will be compared with molecular dynamics simulations. These studies will give unprecedented insights in the mechanisms by which AFPs and AFGPs lower the freezing point of water.