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Protein Engineering vol. 4 no. 8 pp. 1003-1008, 1991
© 1991 Oxford University Press

RESEARCH-ARTICLE

Structure and function of an antifreeze polypeptide from ocean pout, Macrozoarces americanus: role of glutamic acid residues in protein stability and antifreeze activity by site-directed mutagenesis

Xiaomao Li and Choy L. Hew1

Research Institute, The Hospital for Sick Children and the Departments of Biochemistry and Clinical Biochemistry, University of Toronto 100 College Street, Toronto, Ontario, Canada M5G 1L5

1To whom correspondence should be addressed at Department of Clinical Biochemistry

The successful expression and purification of the recombinant ocean pout antifreeze polypeptide (rAFP) in Escherichia coli have enabled the study of its structure-function relationship by site-directed mutagenesis. The role of carboxyl groups at Glu23 and Glu36 of the rAFP was probed by replacing these residues with either glutamine or alanine residues as both single and double mutants. The AFP mutants were expressed, purified and characterized in terms of primary and secondary structures, thermal stability and antifreeze activities. The properties of these mutants were compared with those of the rAFP. Three distinct functions are identified for the carboxyl groups: (i) the negative charges at positions 23 and 36 are involved in the thermal stability of the polypeptide; (ii) the negative charges at positions 23 and 36 contribute to the thermal hysteretic activities of the polypeptide; and (iii) the negative charge at position 23 and hydrogen-bonding ability at position 36 contribute to the ice-binding activity of the polypeptide.

Keywords: ice crystal growth/thermal hysteresis/thermal stability

Received October 5, 1990; accepted August 1, 1991.


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