Folding and structural characterization of highly disulfide-bonded beetle antifreeze protein produced in bacteria

The hyperactive antifreeze protein from the beetle, Tenebrio molitor, is an 8.5-kDa, threonine-rich protein containing 16 Cys residues, all of which a re involved in disulfide bonds. When produced by Escherichia coli, the prot ein accumulated in the supernatant in an inactive, unfolded state. Its corr ect folding. required days or weeks of oxidation at 22 or 4 degrees C, resp ectively, and its purification included the removal of imperfectly folded f orms by reversed-phase HPLC. NMR spectroscopy was used to assess the degree of folding of each preparation. One-dimensional H-1 and two-dimensional H- 1 total correlation spectroscopy spectra were particularly helpful in estab lishing the characteristics of the fully folded antifreeze in comparison to less well-folded forms. The recombinant antifreeze had no free -SH groups and was rapidly and completely inactivated by 10 mM DTT. It had a thermal h ysteresis activity of 2.5 degrees C at a concentration of 1 mg/ml, whereas fish antifreeze proteins typically show a thermal hysteresis of similar to 1.0 degrees C at 10-20 mg/ml. The circular dichroism spectra of the beetle antifreeze had a superficial resemblance to those of alpha-helical proteins , but deconvolution of the spectra indicated the absence of alpha-helix and the presence of beta-structure and coli. NMR analysis and secondary struct ure predictions agree with the CD data and are consistent with a beta-helix model proposed for the antifreeze on the basis of its 12-amino-acid repeat ing structure and presumptive disulfide bond arrangement. (C) 2000 Academic Press.