Isomorphism, cold-adaptation, and phosphorylation of sarcomeric tropomyosin

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

Characterization of Daboia russelii and Naja naja venom neutralizing ability of an undocumented indigenous medication in Sri Lanka

Background: Indigenous medicinal practice in Sri Lanka talks about powerful compounds extracted from native plants for treating venomous snake bites which are hardly documented in literature but are used by the indigenous doctors for thousand years. Objective: We screened the neutralizing ability of a herbal preparation practiced in indigenous medicine of Sri Lanka, consisting of Sansevieria cylindrica, Jatropha podagrica and Citrus aurantiifolia, for its ability to neutralize venom toxins of Naja naja (Common Cobra) and Daboia russelii (Russell's viper). Materials and methods: The venom toxicity was evaluated using a 5-day old chicken embryo model observing the pathophysiology and the mortality for six hours, in the presence or absence of the herbal preparation. The known toxin families to exist in snake venom, such as Phospholipase A2, Snake venom Metalloprotease, were evaluated to understand the mechanism of venom neutralizing ability of the herbal preparation. Results: The LD50 of D. russelii venom, as measured using the 5-day old chicken embryo model, was 4.8 ± 0.865 ug (R2 = 84.8%, P = 0.079). The pre-incubation of venom with the herbal preparation increased the LD50 of D. russelii venom to 17.64 ± 1.35 μg (R2 = 81.0%, P = 0.100), showing a clear neutralizing action of D. russelii venom toxicity by the herbal medicine. Whereas the pre-incubation of venom with the 1× venom neutralizing dose of commercially available polyvalent anti-venom serum shifted the LD50 venom only up to 5.5 ± 1.35 μg (R2 = 98.8%, P = 0.069). In the presence of the herbal preparation, Phospholipase A2 activity of D. russelii venom was significantly reduced from 9.2 × 10−3 mM min−1 to 8.0 × 10−3 mM min−1 and that of N. naja from 2.92 × 10−2 mM min−1 to 0.188 × 10−2 mM min−1. Further, the pre-incubation of N. naja venom with the herbal preparation significantly reduced its Metalloprotease activity from 0.069 units min−1 to 0.019 units min−1. Conclusion: The herbal preparation shows a clear neutralizing action against the toxicities of D. russelii and N. naja venoms demonstrating the potential to be used as a plant based antidote for snake envenomation