Preparation Of A Dendroides Canadensis Antifreeze Protein Mutant

Antifreeze proteins (AFPs) inhibit the growth and recrystallization of ice by binding to small ice crystals. This lowers the freezing point of water without affecting the melting point, resulting in a difference between the two points, termed thermal hysteresis (TH). In previous studies, the focus was identifying which amino acids were important based on the interactions between amino acids, suggested by the three dimensional model of antifreeze protein from the beetle Dendroides canadensis (DAFP-1). It was known that certain low molecular weight molecules can further enhance the antifreeze activity, and certain amino acids, such as arginine, in DAFP-1 are important for the enhanced TH activity in the presence of these small molecules. In general, the degree of TH is dependent upon the specific activity, presence of enhancers, and the concentration of the AFP.  In this study, a novel mutant of DAFP-1, DAFP-1 G49C S-Tag, has been prepared. The mutant will be used to investigate the effect of low molecular weight molecules, which may increase the localized concentration of DAFP-1 G49C S-Tag by interacting with a free cysteine in the S-Tag, on the TH activity. TH activities of DAFP-1 and its mutant in the presence of these small molecules, and alone, will be measured using differential scanning calorimetry.