Atomic-Level Insights into a Unique Semi-Clathrate Hydrate Formed in a Confined Environment of Porous Protein Crystal

Abstract

Water behavior on protein surfaces influences the protein structure and function. Antifreeze proteins (AFPs) have been intensively studied in the context of biological cytotechnology. AFPs can inhibit the growth of ice microcrystals by forming unique water cluster networks that are influenced by protein surface morphology and hydrophobicity. One such unique water cluster network has been identified as semi-clathrate structures in crystals and is believed to be stabilized by intermolecular interactions within the confined environment. However, there is little atomic-level information about the process of formation of semi-clathrates and the structural units of water-clathrate networks. We identified a single semi-clathrate formed on the pore surface of ferritin crystal, which has a structure similar to that of a natural AFP. Comparison of ferritin mutants and determination of temperature-dependent structures revealed that semi-clathrate water molecules on an α-helix undergo structural alterations with increasing temperatures. Lowering the temperature regenerates the semi-clathrate structure. Water molecules hydrogen-bonded to main chain carbonyl groups are stably immobilized at room temperature and serve as starting points for clathrate formation. These findings provide a mechanistic understanding of water networks in AFPs and guidelines for designing new cryomaterials.