De novo designed ice-binding proteins from twist-constrained helices

Attaining molecular-level control over solidification processes is a crucial aspect of materials science. To control ice formation, organisms have evolved bewildering arrays of ice-binding proteins (IBPs) but these have poorly understood structure-activity relationships. We propose that reverse engineering using de novo computational protein design can shed light on structureactivity relationships of IBPs. We hypothesized that the model alpha-helical winter flounder antifreeze protein (wfAFP) uses an unusual under-twisting of its alpha-helix to align its putative ice-binding threonine residues in exactly the same direction. We test this hypothesis by designing a series of straight three-helix bundles with an ice-binding helix projecting threonines and two supporting helices constraining the twist of the ice-binding helix. We find that ice recrystallization inhibition by the designed proteins increases with the degree of designed under-twisting, thus validating our hypothesis and opening up new avenues for the computational design of icebinding proteins. Significance Statement Ice-binding proteins (IBPs) modulate ice nucleation and growth in cold-adapted organisms so that they can survive in ice-laden environments at (sub)freezing temperatures. The functional repertoire of IBPs is diverse, ranging from inhibition of recrystallization and freezing point depression to shaping of ice crystals and ice nucleation. Precisely how these activities arise from the structure and ice-binding properties of IBPs is poorly understood. We demonstrate through de novo computational protein design that constraining the twist of an ice-binding helix is a key feature determining its ice-binding activity, opening new avenues for the design of synthetic IBPs with activities tailored to the requirements of specific applications, such as cell and tissue cryopreservation.