Myopathic mutations in the β‐chain of tropomyosin differently affect the structural and functional properties of ββ‐ and αβ‐dimers

Abstract

Tropomyosin (Tpm) is an actin-binding protein that plays a vital role in the regulation of muscle contraction. Fast skeletal muscles express 2 Tpm isoforms, α (Tpm 1.1) and β (Tpm 2.2), resulting in the existence of 2 forms of dimeric Tpm molecule: αα-homodimer and αβ-heterodimer. ββ-Homodimer is unstable and absent in the native state, despite which most of the studies of myopathy-relating Tpm mutations have been performed on the ββ-homodimer. Here, we applied different methods to investigate the effects of myopathic mutations R133W and N202K in the β-chain of Tpm on properties of αβ-heterodimers and to compare them with the features of ββ-homodimers with the same mutations. The results show that properties of αβ-Tpm and ββ-Tpm with substitutions in the β-chain differ significantly, and this indicates that the effects of myopathic mutations in the Tpm β-chain should be studied on the Tpm αβ-heterodimer.-Bershitsky, S. Y., Logvinova, D. S., Shchepkin, D. V., Kopylova, G. V., Matyushenko, A. M. Myopathic mutations in the β-chain of tropomyosin differently affect the structural and functional properties of ββ- and αβ-dimers.

Congenital myopathy‐related mutations in tropomyosin disrupt regulatory function through altered actin affinity and tropomodulin binding

“…In muscle fibers of patients affected by myopathy‐causing mutations, homodimers with two substituted chains and heterodimers containing single amino acid substitution in one chain are present. Because properties of Tpm isoforms differ quantitatively and are not additive, effects of mutations on heterodimers cannot be easily predicted on the basis of results obtained for homodimers . In this work, we studied effects of the mutations A4V and R91C on functions of Tpm3.12 homodimers; thus, further studies are required to show how wild‐type Tpm3.12 or Tpm2.2 modulate the effects of the substitutions in heterodimers.…”

Mechanisms of disturbance of the contractile function of slow skeletal muscles induced by myopathic mutations in the tropomyosinTPM3gene

“…The affinity of Tpm for actin was estimated using a cosedimentation assay as previously described. 35,36 Briefly, 10 µM of phalloidin stabilized F-actin was mixed with increasing concentrations of Tpm (from 0 to 5-7 µM) at 20°C in 30 mM Hepes-Na (pH 7.3) and 200 mM NaCl to a final volume of 100 µl. After 40 min incubation, actin was pelleted with any bound Tpm by ultracentrifugation at 133 000 g for 40 min (Beckman Airfuge; Beckman Coulter, Fullerton, CA, USA).…”

“…Thermally induced dissociation of Tpm complexes with F-actin stabilized by phalloidin was detected by changes in light scattering at 90 degrees as described earlier. 32,33,[35][36][37] The experiments were performed at 350 nm on a Cary Eclipse fluorescence spectrophotometer (Varian Australia Pty Ltd, Mulgrave, Victoria, Australia) equipped with temperature controller and thermoprobes. All measurements were performed at a constant heating rate of 1°C/min.…”

“…Measurements of the sliding velocity of regulated thin filaments at different Ca 2+ concentrations were performed in the in vitro motility assay as described previously. 1,[34][35][36] In brief, myosin (200 µg/mL) in AB buffer (25 mM KCl, 25 mM imidazole, 4 mM MgCl 2 , 1 mM EGTA, and 20 mM DTT, pH 7.5) containing 0.5 M KCl was loaded into an experimental flow cell with the nitrocellulose-coated inner surface. After 2 min, 0.5 mg/mL BSA was added for 1 min.…”

Influence of tropomodulin 1 on calcium regulation of actin-myosin interaction in the ventricles

“…Methods: Myosin and native thin filaments (NTF) consisting of actin, troponin, and tropomyosin were isolated from the left ventricle of sheep. Human Tmod1 was expressed in E. coli [4]. To study the effect of Tmod1 on actin-myosin interaction we used an in vitro motility assay [4].…”