Deciphering the Role of the Non-ice-binding Surface in the Antifreeze Activity of Hyperactive Antifreeze Proteins.

Antifreeze proteins (AFPs) show thermal hysteresis through specific interaction with the ice crystal. Hyperactive AFPs interact with the ice surface through a threonine-rich motif present at their ice-binding surface (IBS). Ordering of water around the IBS was extensively investigated. However, the role of non-IBS in ice growth inhibition is yet to be understood completely. The present study explores the nature of hydration and its length-scale evaluation around the non-IBS for hyperactive AFPs. We observed that the hydration layer of non-IBS is liquid-like, even in highly supercooled conditions, and the nature of hydration is drastically different from the hydration pattern of non-AFP surfaces. In similar conditions, the hydration layer around the IBS is ice-like ordered. Non-IBS of the hyperactive AFP exposes toward the bulk and is able to maintain the liquid-like character of its hydration water up to 15 Å. We also find that the amino acid compositions and their spatial distribution on the non-IBS are markedly different from those of the IBS and non-AFP surfaces. These results elucidate the combined role of IBS and non-IBS in ice-growth inhibition. While IBS is required to adsorb on ice efficiently, the exposed non-IBS may prevent ice nucleation/growth on top of the bound AFPs.