UC Irvine

Effects of a beetle antifreeze proteins (AFP) from Dendroides canadensis (DAFP-1) on a model freeze-labile enzyme, lactate dehydrogenase (LDH), were investigated under freezing and thawing conditions. The presence of DAFP-1 can effectively protect the enzymatic activity of LDH upon repeated freezing and thawing and the protective role of DAFP-1 is more significant than that of bovine serum albumin (BSA), a common protectant for freeze-labile proteins. The results of circular dichroism (CD) spectroscopy suggest that the presence of DAFP-1 provides protection to the denaturation of LDH under freezing and thawing. The molecular dynamics (MD) simulation of DAFP-1 and LDH suggests that DAFP-1 interacts with LDH using its ice-binding surface (IBS) and mainly through its arginine residues. A mutant of DAFP-1, where all the arginine residues were replaced by alanine residues, lost its effect in protecting LDH under freezing and thawing. The results demonstrated that DAFP-1 is an effective protectant for a freeze-labile protein under freezing and thawing and the arginine residues in DAFP-1 are important for its protective role. By correlating the protective effect of an AFP with its structure, new insights in the identification and development of effective protectants for freeze-labile proteins were provided.