Novel dimeric β-helical model of an ice nucleation protein with bridged active sites

Here we have predicted a novel β-helical fold for the INP produced by the bacterium Pseudomonas borealis. The protein uses internal serine and glutamine ladders for stabilization and is predicted to dimerize via the burying of a solvent-exposed tyrosine ladder to make an intimate hydrophobic contact along the dimerization interface. The manner in which PbINP dimerizes also allows for its multimerization, which could explain the aggregation-dependence of INP activity. Both sides of the PbINP structure have tandem arrays of amino acids that can organize waters into the ice-like clathrate structures seen on antifreeze proteins.Dimerization dramatically increases the 'ice-active' surface area of the protein by doubling its width, increasing its length, and presenting identical ice-forming surfaces on both sides of the protein. We suggest that this allows sufficient anchored clathrate waters to align on the INP surface to nucleate freezing. As PbINP is highly similar to all known bacterial INPs, we predict its fold and mechanism of action will apply to these other INPs.Two extraordinary families of proteins have evolved to influence ice growth in opposite ways: antifreeze proteins (AFPs) that irreversibly adsorb to the surface of ice crystals to prevent their further growth [1]; and ice-nucleation proteins (INPs) that cause ice to form in solution at high sub-zero temperatures [2,3]. Whereas AFPs are small (Mr 3,000 - 35,000), and generally monomeric proteins, INPs are large (Mr >100,000) and function as multimers [4].The tertiary structures of many AFPs are known, but none has been experimentally determined for an INP. Most INPs contain three distinct domains, with the majority of their mass residing within a highly repetitive central domain [5]. This domain consists of a variable number (ca. 50-80) of tandem 16-amino-acid (aa) repeats, with each repeat following the general consensus sequence of GYGSTxTAxxxSxLxA [6]. NMR and CD studies of synthetic bacterial INP peptid