Identification of the ice-binding face of antifreeze protein from Tenebrio molitor

AbstractThe beetle Tenebrio molitor produces several isoforms of a highly disulfide-bonded β-helical antifreeze protein with one surface comprised of an array of Thr residues that putatively interacts with ice. In order to use mutagenesis to identify the ice-binding face, we have selected an isoform that folds well and is tolerant of amino acid substitution, and have developed a heating test to monitor refolding. Three different types of steric mutations made to the putative ice-binding face reduced thermal hysteresis activity substantially while a steric mutation on an orthogonal surface had little effect. NMR spectra indicated that all mutations affected protein folding to a similar degree and demonstrated that most of the protein folded well. The large reductions in activity associated with steric mutations in the Thr array strongly suggest that this face of the protein is responsible for ice binding