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 proteins (AFPs) at depressing solution freezing points via ice-crystal growth inhibition. Since this insect protein is only available in microgram quantities from its natural source, recombinant sbwTHP was produced from inclusion bodies in Escherichia coli by a refolding protocol. Incompletely folded forms were removed during ion-exchange and reverse-phase chromatography, resulting in fully active sbwTHP that was indistinguishable in its properties from native sbwTHP. The antifreeze was completely inactivated by reduction, showed no reaction with sulfhydryl reagents, and was not inhibited by EDTA. All 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 eighth, and the sixth and seventh. NMR analysis confirmed that the fully folded form of sbwTHP was well structured and had a single conformation. Both NMR and CD spectra indicate the presence of extensive beta structure (70-80%) with little or no alpha helix. The protein maintains antifreeze activity over a broad range of pH values, and its conformation is independent of both temperature (over the range 0 degrees C to 20 degrees C), and the presence of 50% trifluoroethanol.
Gauthier
S Y
SY
Department of Biochemistry, Queen's University, Kingston, Ontario, Canada.
Kay
C M
CM
Sykes
B D
BD
Walker
V K
VK
Davies
P L
PL
D000595:Amino Acid Sequence; D000818:Animals; D021301:Antifreeze Proteins; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry*; D006023:Glycoproteins / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D019476:Insect Proteins / Q000737:chemistry; D007915:Lepidoptera / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D017421:Sequence Analysis; D014357:Trypsin / Q000378:metabolism
D021301:Antifreeze Proteins; D004220:Disulfides; D006023:Glycoproteins; D019476:Insect Proteins; D011994:Recombinant Proteins; D014357:Trypsin