The Role of Ca2+-Coordinating Residues of Herring Antifreeze Protein in Antifreeze Activity

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Journal titleBiochemistry
Pages1454714554; # of pages: 8
Subjectantifreeze activity; Atlantic herring; Clupea harengus harengus
AbstractThe type II antifreeze protein of Atlantic herring (Clupea harengus harengus) requires Ca2+ as a cofactor to inhibit the growth of ice crystals. On the basis of homology modeling with Ca2+-dependent lectin domains, five residues of herring antifreeze protein (hAFP) are predicted to be involved in Ca2+ binding: Q92, D94, E99, N113, and D114. The role of E99, however, is less certain. A previous study on a double mutant EPN of hAFP suggested that the Ca2+-binding site of hAFP was the ice-binding site. However, it is possible that Ca2+ might function distantly to affect ice binding. Site-directed mutagenesis was performed on the Ca2+-coordinating residues of hAFP in order to define the location of the icebinding site and to explore the role of these residues in antifreeze activity. Properties of the mutants were investigated in terms of their structural integrity and antifreeze activity. Equilibrium dialysis analysis demonstrated that E99 is a Ca2+-coordinating residue. Moreover, proteolysis protection assay revealed that removal of Ca2+ affected the conformation of the Ca2+-binding loop rather than the core structure of hAFP. This finding rules out the possibility that Ca2+ might act at a distance via a conformational change to affect the function of hAFP. Substitutions at positions 99 and 114 resulted in severely reduced thermal hysteresis activity. These data indicate that the ice-binding site of hAFP is located at the Ca2+-binding site and the loop region defined by residues 99 and 114 is important for antifreeze activity.
Publication date
PublisherACS Publications
Copyright noticeCopyright 2004 American Chemical Society
AffiliationNRC Institute for Marine Biosciences; National Research Council Canada
Peer reviewedYes
NRC number1690
NPARC number3538344
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Record identifier595bccbd-72b1-421e-8b00-8befb906f26b
Record created2009-03-01
Record modified2016-05-09
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