Type I shorthorn sculpin antifreeze protein: recombinant synthesis, solution conformation, and ice growth inhibition studies.
A number of structurally diverse classes of "antifreeze" proteins that allow fish to survive in sub-zero ice-laden waters have been isolated from the blood plasma of cold water teleosts. However, despite receiving a great deal of attention, the one or more mechanisms through which these proteins act are not fully understood. In this report we have synthesized a type I antifreeze polypeptide (AFP) from the shorthorn sculpin Myoxocephalus scorpius using recombinant methods. Construction of a synthetic gene with optimized codon usage and expression as a glutathione S-transferase fusion protein followed by purification yielded milligram amounts of polypeptide with two extra residues appended to the N terminus. Circular dichroism and NMR experiments, including residual dipolar coupling measurements on a 15N-labeled recombinant polypeptide, show that the polypeptides are alpha-helical with the first four residues being more flexible than the remainder of the sequence. Both the recombinant and synthetic polypeptides modify ice growth, forming facetted crystals just below the freezing point, but display negligible thermal hysteresis. Acetylation of Lys-10, Lys-20, and Lys-21 as well as the N terminus of the recombinant polypeptide gave a derivative that displays both thermal hysteresis (0.4 degrees C at 15 mg/ml) and ice crystal faceting. These results confirm that the N terminus of wild-type polypeptide is functionally important and support our previously proposed mechanism for all type I proteins, in which the hydrophobic face is oriented toward the ice at the ice/water interface.
Fairley
Kayesh
K
School of Chemistry and the Department of Biochemistry, University of Sydney, New South Wales 2006, Australia.
Westman
Belinda J
BJ
Pham
Linda H
LH
Haymet
A D J
AD
Harding
Margaret M
MM
Mackay
Joel P
JP
D000595:Amino Acid Sequence; D000818:Animals; D021301:Antifreeze Proteins / Q000737:chemistry* / Q000235:genetics; D001483:Base Sequence; D002942:Circular Dichroism; D017931:DNA Primers; D029941:Fish Proteins; D005813:Genes, Synthetic; D007053:Ice; D009682:Magnetic Resonance Spectroscopy; D018715:Microscopy, Video; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis; D012605:Scorpions; D012996:Solutions
D021301:Antifreeze Proteins; D017931:DNA Primers; D029941:Fish Proteins; D007053:Ice; D011994:Recombinant Proteins; D012996:Solutions; C446459:antifreeze protein, Myoxocephalus scorpius