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 are involved in disulfide bonds. When produced by Escherichia coli, the protein accumulated in the supernatant in an inactive, unfolded state. Its correct folding required days or weeks of oxidation at 22 or 4 degrees C, respectively, and its purification included the removal of imperfectly folded forms by reversed-phase HPLC. NMR spectroscopy was used to assess the degree of folding of each preparation. One-dimensional (1)H and two-dimensional (1)H total correlation spectroscopy spectra were particularly helpful in establishing 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 hysteresis activity of 2.5 degrees C at a concentration of 1 mg/ml, whereas fish antifreeze proteins typically show a thermal hysteresis of approximately 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 coil. NMR analysis and secondary structure 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 repeating structure and presumptive disulfide bond arrangement.
Liou
Y C
YC
Department of Biochemistry, Queen's University, Kingston, Ontario, Canada.
Daley
M E
ME
Graham
L A
LA
Kay
C M
CM
Walker
V K
VK
Sykes
B D
BD
Davies
P L
PL
D000818:Animals; D021301:Antifreeze Proteins; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D001517:Coleoptera / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006023:Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D009682:Magnetic Resonance Spectroscopy; D010084:Oxidation-Reduction; D010449:Peptide Mapping; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013912:Threonine / Q000737:chemistry; D014357:Trypsin / Q000494:pharmacology
D021301:Antifreeze Proteins; D004220:Disulfides; D006023:Glycoproteins; D011994:Recombinant Proteins; D013912:Threonine; D014357:Trypsin; D003545:Cysteine