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Originally published In Press as doi:10.1074/jbc.M200307200 on April 8, 2002
J. Biol. Chem., Vol. 277, Issue 27, 24073-24080, July 5, 2002
Type I Shorthorn Sculpin Antifreeze Protein
RECOMBINANT SYNTHESIS, SOLUTION CONFORMATION, AND ICE
GROWTH INHIBITION STUDIES*
Kayesh
Fairley §,
Belinda J.
Westman§¶,
Linda H.
Pham ,
A. D. J.
Haymet**,
Margaret M.
Harding, and
Joel P.
Mackay§§§
From the School of Chemistry and the
§ Department of Biochemistry, University of Sydney, New
South Wales 2006, Australia and the Department of Chemistry,
University of Houston, Houston, Texas 77204-5003
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  -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 °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.
*
This research was supported in part by a University of
Sydney Sesquicentennial Research and Development Grant (to M. M. H.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
**
Supported by the Welch Foundation and Grant ARP
003652- 0303-1999.
¶
Supported by an Australian Postgraduate Award.
To whom correspondence may be addressed: The School of
Chemistry, University of Sydney, New South Wales 2006, Australia. Tel.: 61-2-9351-2745; Fax: 61-2-9351-6650; E-mail:
harding@chem.usyd.edu.au.
§§
An ARC Research Fellow. To whom correspondence may be addressed:
The Department of Biochemistry, University of Sydney, New South Wales
2006, Australia. Tel.: 61-2-9351-3906; Fax: 61-2-9351-4726; E-mail: j.mackay@biochem.usyd.edu.au.
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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Copyright © 2002 by the American Society for Biochemistry and Molecular Biology.
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