The Role of Ca<sup>2+</sup>-Coordinating Residues of Herring Antifreeze Protein in Antifreeze Activity
Bibliographic record
Abstract
The type II antifreeze protein of Atlantic herring (Clupea harengus harengus) requires Ca(2+) as a cofactor to inhibit the growth of ice crystals. On the basis of homology modeling with Ca(2+)-dependent lectin domains, five residues of herring antifreeze protein (hAFP) are predicted to be involved in Ca(2+) 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 Ca(2+)-binding site of hAFP was the ice-binding site. However, it is possible that Ca(2+) might function distantly to affect ice binding. Site-directed mutagenesis was performed on the Ca(2+)-coordinating residues of hAFP in order to define the location of the ice-binding 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 Ca(2+)-coordinating residue. Moreover, proteolysis protection assay revealed that removal of Ca(2+) affected the conformation of the Ca(2+)-binding loop rather than the core structure of hAFP. This finding rules out the possibility that Ca(2+) 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 Ca(2+)-binding site and the loop region defined by residues 99 and 114 is important for antifreeze activity.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".