Cold adaptation of Atlantic salmon tropomyosin: role of residue '77'
Notice bibliographique
Résumé
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.
Récupéré en direct depuis OpenAlex et désinversé. Les résumés ne sont pas conservés dans cette base de données : les index inversés représentent 8,6 Go des 9,3 Go de texte de la base, et le serveur dispose de 13 Go libres.
Comment cette classification a été obtenuedéplier
Prédiction machine sur la base complète
Imitation des enseignantsNi prévalence calibrée, ni vérité terrain. Validation humaine à venir. Le volet Gemma est une étiquette directe du modèle pour chaque travail de la base, lue sur la notice réduite au titre. Le volet Codex est un classifieur appris des 10 348 étiquettes directes de Codex et calibré sur les taux pondérés de l'échantillon; les champs sans appui suffisant ne portent aucun appel Codex. Le mode candidate est l'union des deux volets; le consensus est leur intersection. Ces sorties portent le statut machine_predicted_unvalidated et ne sont pas des étiquettes humaines.
Scores du classifieur distillé par catégorie (deux têtes)
| Catégorie | Codex | Gemma |
|---|---|---|
| Métarecherche | 0,000 | 0,000 |
| Méta-épidémiologie (sens strict) | 0,000 | 0,000 |
| Méta-épidémiologie (sens large) | 0,000 | 0,000 |
| Bibliométrie | 0,000 | 0,000 |
| Études des sciences et des technologies | 0,000 | 0,000 |
| Communication savante | 0,000 | 0,000 |
| Science ouverte | 0,000 | 0,000 |
| Intégrité de la recherche | 0,000 | 0,000 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,001 | 0,000 |
Scores machine (provisoires)
Les deux têtes enseignantes du modèle étudiant, lues sur ce travail. Un score ordonne la base pour la relecture; il n'affirme jamais une catégorie, et le statut de validation accompagne chaque rangée tel quel.
Scores de référence d'un modèle non mature (critères de maturité non atteints, 7 itérations). Un score ordonne; il n'affirme jamais une catégorie.
score_only:v0-immature-baseline · tel quel depuis la passe de notation : score_only signifie que le nombre peut ordonner les travaux, et qu'aucune étiquette de catégorie n'en découleClassification
machine, non validéePrédiction automatique; un appel candidat d’une seule source (Gemma direct ou Codex distillé), pas un consensus.
Le détail, modèle par modèle et score par score, se trouve en fin de page sous « Comment cette classification a été obtenue ».