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.
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.
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Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada.
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D000409:Alanine / Q000031:analogs & derivatives; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D021301:Antifreeze Proteins; D001483:Base Sequence; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics; D005399:Fishes / Q000235:genetics*; D005615:Freezing; D015964:Gene Expression Regulation, Bacterial; D005971:Glutamates / Q000737:chemistry; D018698:Glutamic Acid; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D007053:Ice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013329:Structure-Activity Relationship
D000596:Amino Acids; D021301:Antifreeze Proteins; D005971:Glutamates; D006023:Glycoproteins; D007053:Ice; D011994:Recombinant Proteins; D018698:Glutamic Acid; D000409:Alanine