Thermodynamic stability of a cold‐adapted protein, type III antifreeze protein, and energetic contribution of salt bridges
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Bibliographic record
Abstract
A thermodynamic analysis of a cold-adapted protein, type III anti-freeze protein (AFP), was carried out. The results indicate that the folding equilibrium of type III AFP is a reversible, unimolecular, two-state process with no populated intermediates. Compared to most mesophilic proteins whose folding is two-state, the psychrophilic type III AFP has a much lower thermodynamic stability at 25 degrees C, approximately 3 kcal/mol, and presents a remarkably downshifted stability-temperature curve, reaching a maximum of 5 kcal/mol around 0 degrees C. Type III AFPs contain few and non-optimally distributed surface charges relative to their mesophilic homologs, the C-terminal domains of sialic acid synthases. We used thermodynamic double mutant cycles to evaluate the energetic role of every surface salt bridge in type III AFP. Two isolated salt bridges provided no contribution to stability, while the Asp36-Arg39 salt bridge, involved in a salt bridge network with the C-terminal carboxylate, had a substantial contribution (approximately 1 kcal/mol). However, this contribution was more than counteracted by the destabilizing effect of the Asp36 carboxylate itself, whose removal led to a net 30% increase in stability at 25 degrees C. This study suggests that type III AFPs may have evolved for a minimally acceptable stability at the restricted, low temperature range (around 0 degrees C) at which AFPs must function. In addition, it indicates that salt bridge networks are used in nature also for the stability of psychrophilic proteins, and has led to a type III AFP variant of increased stability that could be used for biotechnological purposes.
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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 | 0.001 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.001 |
| Science and technology studies | 0.000 | 0.001 |
| Scholarly communication | 0.000 | 0.001 |
| 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 it