The fold of the β-helical antifreeze protein from Tenebrio molitor (TmAFP) proved to be surprisingly tolerant of multiple amino acid substitutions, enabling the construction of a panel of mutants displaying grids of single amino acid types in place of the threonines on the ice-binding face. These mutants, maintaining the regularity of amino acid spacing found in the wild-type protein but with different functional groups on the surface, were tested for antifreeze activity by measuring thermal hysteresis and observing ice grown in their presence. We found that no mutant exhibited the dramatic activity of the wild-type version of this hyperactive antifreeze protein. However, mutants containing four valines or tyrosines in place of the threonines in the center of the TmAFP ice-binding face showed residual thermal hysteresis activity and had marked effects on ice crystal morphology. The results are discussed in the context of a two-stage model for the absorption-inhibition mechanism of antifreeze protein binding to ice surfaces.