Water entropy at the threonine-rich surface of antifreeze and ice-nucleating proteins: small changes make a big difference

Heterogeneous ice nucleation is vital for various natural processes and for maintaining global sea levels. Although different ice nucleating proteins (INPs) have been discovered on naturally formed ice, many organisms possess antifreeze proteins (AFPs) which are structurally similar to INPs and yet prevent ice growth in their body fluid. In this study, we investigate the ice nucleation efficiency of INPs over AFPs by looking into water entropy near these proteins. Using all-atom molecular dynamics simulations and a method to calculate the entropy of individual water molecules, we found distinct water entropy patterns near AFPs and INPs. For the INP structure, water molecules hydrogen-bonded to threonine residues on the ice-binding surface (IBS) exhibited the most significant entropy decrease, likely lowering the entropic barrier for ice nucleation. Even for water near the IBS of the two AFPs studied, the entropy patterns have been found to be dissimilar. Our analysis reveals that the entropy patterns stem from varying fluctuation levels of threonine side chains on the IBS. Consequently, for two INP conformations differing in the orientation of a conserved loop near the IBS, one structure has been found not to lower water entropy as effectively as the other. Our study reveals that larger surface areas or continuous threonine patches are not the only criteria that create differences between AFPs and INPs. The extent of rigidity and consequently the change in water entropy behavior enhances the ice nucleation efficiency of INPs under moderate supercooling conditions.