Preferential Ordering and Organization of Hydration Water Favor Nucleation of Ice by Ice-Nucleating Proteins over Antifreeze Proteins.

Bacteria containing ice-nucleating proteins (INPs) evolved in nature to nucleate ice at the high sub-zero ambiance. The ability of the INPs to induce order in the hydration layer and their aggregation propensity appear to be key factors of their ice nucleation abilities. However, the mechanism of the process of ice nucleation by INPs is yet to be understood clearly. Here, we have performed all-atom molecular dynamics simulations and analyzed the structure and dynamics of the hydration layer around the proposed ice-nucleating surface of a model INP. Results are compared with the hydration of a topologically similar non-ice-binding protein (non-IBP) and another ice-growth inhibitory antifreeze protein (sbwAFP). We observed that the hydration structure around the ice-nucleating surface of INP is highly ordered and the dynamics of the hydration water are slower, compared to the non-IBP. Even the ordering of the hydration layer is more evident around the ice-binding surface of INP, compared to the antifreeze protein sbwAFP. Particularly with increasing repeat units of INP, we observe an increased population of ice-like water. Interestingly, the distances between the hydroxyl groups of the threonine ladder and its associated channel water of the ice-binding surface (IBS) of INP in the X and Y direction mimic the oxygen atom distances of the basal plane of hexagonal ice. However, the structural synergies between the hydroxyl group distances of the threonine ladder and its associated channel water of the IBS of sbwAFP and oxygen atom distances of the basal plane are less evident. This difference makes the IBS of the INP a better template for ice nucleation than AFP, although both of them bind to the ice surface efficiently.

Molecular Factors of Ice Growth Inhibition for Hyperactive and Globular Antifreeze Proteins: Insights from Molecular Dynamics Simulation.

The molecular mechanism behind the ice growth inhibition by antifreeze proteins (AFPs) is yet to be understood completely. Also, what physical parameters differentiate between the AFP and non-AFP are largely unknown. Thus, to get an atomistic overview of the differential antifreeze activities of different classes of AFPs, we have studied ice growth from different ice surfaces in the presence of a moderately active globular type III AFP and a hyperactive spruce budworm (sbw) AFP. Results are compared with the observations of ice growth simulations in the presence of topologically similar non-AFPs using all-atom molecular dynamics simulations. Simulation data suggest that the ice surface coverage is a critical factor in ice growth inhibition. Due to the presence of an ice binding surface (IBS), AFPs form a high affinity complex with ice, accompanied by a transition of hydration water around the IBS from clathrate-like to ice-like. Several residues around the periphery of the IBS anchor the AFP to the curved ice surface mediated by multiple strong hydrogen bonds, stabilizing the complex immensely. In the high surface coverage regime, the slow unbinding kinetics dominates over the ice growth kinetics and thus facilitates the ice growth inhibition. Due to the non-availability of a proper IBS, non-AFPs form a low-affinity complex with the growing ice surface. As a result, the non-AFPs are continuously repelled by the surface. If the concentration of AFPs is low, then the effective surface coverage is reduced significantly. In this low surface coverage regime, AFPs can also behave like impurities and are engulfed by the growing ice crystal.