Isomorphism, cold-adaptation, and phosphorylation of sarcomeric tropomyosin

Silva, A. Madhushika M. (2021) Isomorphism, cold-adaptation, and phosphorylation of sarcomeric tropomyosin. Doctoral (PhD) thesis, Memorial University of Newfoundland.

[English] PDF - Accepted Version Available under License - The author retains copyright ownership and moral rights in this thesis. Neither the thesis nor substantial extracts from it may be printed or otherwise reproduced without the author's permission. Download (7MB)

Abstract

Tropomyosin is a dimeric protein containing 284 amino acids per chain that is found, in association with F-actin and troponin, within the thin filament, a complex that regulates muscle contraction. The thesis is composed of three results chapters: i) The existence of beta tropomyosin (Tpm2) is demonstrated for the first time in fish (Salmo salar). Compared to the mammalian homologue, salmon Tpm2 has fewer cysteine (one per chain) and tyrosine (five per chain) residues. Tpm2 contributes half of the total tropomyosin in the jaw, tongue, and fin muscles. A Tpm1 isoform, either alpha-1 chain-like isoform X1 (cheek dark, jaw and tongue) or alpha (fin), accounts for the remainder. Salmon tropomyosins are distinguishable based on electrophoretic mobility, affinity for troponin-Sepharose, tryptic peptide mapping, and variable carboxyl-terminal regions (residues 276 - 284): beta (Tpm2), Leu-Ala-Leu-Asn-Asp-Met-Thr-Thr-Leu; alpha-1 chain-like isoform X1, His-Ala-Leu-Asn-Asp-Met-Thr-Ala-Ile, and alpha (Tpm1), Asn-Ala-Leu-Asn-Asp-Met-Thr-Ser-Ile. ii) The relative instability of the most abundant isoform of Atlantic salmon, Tpm1, (20 substitutions vs. mammal) is due to a neutral 77th amino acid (Thr in salmon; Lys in rabbit), and glycines at 24 and 27 (Ala and Gln in rabbit). Incorporation of the respective mesophilic amino acid increases resistance to thermal unfolding as determined by calorimetry and chymotrypsin digestion at Leu-169, a site ⁓100 amino acids far away from residue-77. PyMOL indicates ion pairing between Lys-77 and Glu-82 in the opposite chain. Binding of Tpm1 to troponin-Sepharose is influenced by N-terminal acetylation and the mutation of residues 24, 27, and 77. Furthermore, wild type Tmp1 displays a higher affinity for F-actin at 4 ℃ (KD, ̴ 0.1 μM) than 30 ℃ (KD, ̴ 1.6 μM). In contrast, the mesophilic homologue binds less tightly to actin at lower temperatures. iii) Phosphorylation of serine 283 increases the susceptibility of mammalian Tpm1.1(α) to chymotrypsin (at Leu-169) and trypsin (at Arg-133), suggesting an induced opening of the center of the molecule >150 amino acids upstream, and shifts the corresponding portion of the circular dichroism unfolding profile. These results infer a change in exposure of actin-binding periods 4 (residues 124 - 147) and 5 (residues 166 - 189). The proposal is consistent with a two-fold increase in affinity for F-actin in co-sedimentation experiments.

Actions (login required)

View Item