Solution structure of a recombinant type I sculpin antifreeze protein.

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 K, Westman BJ, Pham LH, Haymet AD, Harding MM, Mackay JP.
Type I shorthorn sculpin antifreeze protein: recombinant synthesis, solution
conformation, and ice growth inhibition studies.
J Biol Chem. 2002 Jul 5;277(27):24073-80. Epub 2002 Apr 08.

Type I shorthorn sculpin antifreeze protein: recombinant synthesis, solution conformation, and ice growth inhibition studies.

simulated annealing, molecular dynamics, torsion angle dynamics

Bartels, C.; Xia, T.-H.; Billeter, P.; Guntert, P.; Wuthrich, K.
J. Biomol. NMR 1995, 5, 1-10.

Goddard, T.D. and Kneller, D.G. SAPRKY 3,
University of California, San Francisco, USA.

Nilges, M. Fold Des 1997, 2, S53-57.
Linge, J. P.; O'Donoghue, S. I.; Nilges, M. Meth. Enzymol. 2001, 339, 71-90.
Nilges, M.; Macias, M. J.; O'Donoghue, S. I.; Oschkinat, H. J. Mol. Biol. 1997, 269, 408-422.
Nilges, M. J. Mol. Biol. 1995, 245, 645-660.