Bibliographic record
Abstract
Tropomyosin from the fast skeletal (trunk) muscle of Atlantic salmon (Salmo salar) is an alpha-type isoform. It shares ~93% identity with a counterpart from rabbit skeletal muscle (20 amino acid substitutions out of a total of 284) but is less thermally stable. -- An interesting aspect of the small heterogeneity is the replacement of lysine-77 in rabbit with threonine in salmon. In the case of the mammalian protein, the lysine in question is positioned to form ion pairs with aspartate-80 in the next turn of the same helix and also with glutamate 82 in the opposing helix of the coiled coil. Thus, at neutral pH, salmon tropomyosin loses two potential charge-charge (ion pair) interactions. The contribution of residue-77 to the properties of salmon tropomyosin has been investigated by mutating it to the amino acid in rabbit using site directed mutagenesis, followed by circular dichroism, differential scanning calorimetry, affinity chromatography and limited proteolysis. A major finding of this research is that threonine in the 77th position of salmon tropomyosin is destabilizing compared to lysine in the same position. Reducing the number of ion pairs is concluded to be part of the structural means by which tropomyosin, and possibly other rod-shaped proteins, adapt to low temperature. The specific details are outlined below in point form: -- 1. Atlantic salmon fast skeletal muscle alpha-tropomyosin cDNA was mutated to replace the threonine at position 77 with lysine using the QuickChange Lightning site directed mutagenesis kit. The mutation was confirmed by DNA sequencing. -- 2. Mutated and non mutated recombinant tropomyosins were obtained by expression in E. coli BL21 cells and chromatographically isolated. -- 3. The mutant salmon tropomyosin, containing Lys-77, exhibited a faster electrophoretic mobility than the non-mutant (Thr-77), in the presence of the detergent sodium dodecyl sulfate, which is attributable to a difference in detergent binding due to the extra charge. This electrophoretic shift was useful in the identification of amino-terminal peptides (see below, #7). -- 4. Far-UV circular dichroism was used to investigate the conformational stability of the recombinant salmon tropomyosins (ionic strength 0.1 M, +DTT, pH 7.0). The observed melting temperatures of the mutant (~45°C) and non mutant (~40°C) recombinant proteins differ by approximately 5°C. -- 5. Differential scanning calorimetry was employed to monitor the thermal unfolding of non mutant and mutant recombinant tropomyosin (ionic strength 0.1 M, +DTT, pH 7.0) by direct heating. The observed melting temperatures of the mutant (~44°C) and non mutant (~40.5°C) protein differ by approximately 4°C. -- 6. In an affinity chromatography experiment mutant tropomyosin bound more strongly to troponin-Sepharose 4B than the non mutant. -- 7. The time-course of limited chymotryptic or tryptic digestion (T, 25°C) was monitored by SDS PAGE and found to be similar for mutant and non-mutant tropomyosins. Edman-based sequencing showed that the initial chymotryptic cleavage site in salmon recombinant tropomyosin (both mutant and non-mutant) is between Leu-11 and Lys-12. The second chymotryptic site is between Leu-169 and Val-170 which corresponds to the preferred site reported previously in rabbit alpha-tropomyosin.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| 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) | 0.001 | 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 source (direct Gemma or distilled Codex), 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".