Molecular Insights into the Unusual Structure of an Antifreeze Protein with a Hydrated Core.

The primary driving force for protein folding is the formation of a well-packed, anhydrous core. However, recently, the crystal structure of an antifreeze protein, maxi, has been resolved where the core of the protein is filled with water, which apparently contradicts the existing notion of protein folding. Here, we have performed standard molecular dynamics (MD) simulation, replica exchange MD (REMD) simulation, and umbrella sampling using TIP4P water at various temperatures (300, 260, and 240 K) to explore the origin of this unusual structural feature. It is evident from standard MD and REMD simulations that the protein is found to be stable at 240 K in its unusual state. The core of protein has two layers of semi-clathrate water separating the methyl groups of alanine residues from different helical strands. However, with increasing temperature (260 and 300 K), the stability decreases as the core becomes dehydrated, and methyl groups of alanine are tightly packed driven by hydrophobic interactions. Calculation of the potential of mean force by an umbrella sampling technique between a pair of model hydrophobes resembling maxi protein at 240 K shows the stabilization of second solvent-separated minima (SSM), which provides a thermodynamic rationale of the unusual structural feature in terms of weakening of the hydrophobic interaction. Because the stabilization of SSMs is implicated for cold denaturation, it suggests that the maxi protein is so designed by nature where the cold denatured-like state becomes the biologically active form as it works near or below the freezing point of water.