Disulfide bond mapping and structural characterization of spruce budworm antifreeze protein

The 9-kDa, Thr-, Ser-, and Cys-rich thermal hysteresis protein from spruce budworm (sbwTHP) is 10-30 times more effective than fish antifreeze protein s (AFPs) at depressing solution freezing points via ice-crystal growth inhi bition. Since this insect protein is only available in microgram quantities from its natural source, recombinant sbwTHP was produced from inclusion bo dies in Escherichia coli by a refolding protocol. Incompletely folded forms were removed during ion-exchange and reverse-phase chromatography, resulti ng in fully active sbwTHP that was indistinguishable in its properties from native sbwTHP. The antifreeze was completely inactivated by reduction, sho wed no reaction with sulfhydryl reagents, and was not inhibited by EDTA. Al l eight cysteine residues appear to be involved in disulfide bond formation . Tryptic cleavage and peptide analysis is consistent with linkages between the first and second cysteine residues, the third and fourth, fifth and ei ghth, and the sixth and seventh. NMR analysis confirmed that the fully fold ed form of sbwTHP was well structured and had a single conformation. Both N MR and CD spectra indicate the presence of extensive beta structure (70-80% ) with little or no cc helix. The protein maintains antifreeze activity ove r a broad range of pH values, and its conformation is independent of both t emperature lover the range 0 degrees C to 20 degrees C), and the presence o f 50% trifluoroethanol.