title: Impaired tropomyosin-troponin interactions reduce activation of the actin thin filament.
abstract: Tropomyosin and troponin are bound to the actin filament to control the contraction of striated muscle in the Ca-dependent manner. The interactions between both regulatory proteins important for the regulation process are not fully understood. To gain more insight into the mechanisms of the thin filament regulation by skeletal α-tropomyosin and troponin, we analyzed effects of seven myopathy-related substitutions: Leu99Met, Ala155Thr, Arg167Gly, Arg167Cys, Arg167His, Lys168Glu, and Arg244Gly. All substitutions reduced Ca-dependent activation of the actomyosin ATPase. The effects of mutations in Arg167 and Lys168 were the most severe. The amino acid substitutions did not significantly affect troponin binding to the whole filament, but reduced 1.2-2.8 fold the affinity of troponin to tropomyosin alone. The excimer fluorescence of N-(1-pyrene)iodoacetamide, a probe attached to the central Cys190, demonstrated that substitutions located near the troponin core domain-binding region strongly affected conformational changes accompanying the tropomyosin-troponin interactions. The thermal stability of all tropomyosin mutants was lower than the stability of the wild type tropomyosin, with TM reduced by 5.3-8.5°C. Together the analyses demonstrated that the myopathy-causing mutations affected tropomyosin structure and led to changes in interactions between tropomyosin and troponin, which impaired the transition of the thin filament from the inactive off to the active on state.
authors.affiliation: Department of Biochemistry and Cell Biology, Faculty of Natural Sciences, Kazimierz Wielki University in Bydgoszcz, Chodkiewicz 30, Str., 85-064 Bydgoszcz, Poland.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Robaszkiewicz
authors.affiliation: Department of Biochemistry and Cell Biology, Faculty of Natural Sciences, Kazimierz Wielki University in Bydgoszcz, Chodkiewicz 30, Str., 85-064 Bydgoszcz, Poland.
authors.forename: Zofia
authors.initials: Z
authors.lastname: Ostrowska
authors.affiliation: BLIRT S.A., Gdańsk, Trzy Lipy 3/1.38, 80-172, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Cyranka-Czaja
authors.affiliation: Department of Biochemistry and Cell Biology, Faculty of Natural Sciences, Kazimierz Wielki University in Bydgoszcz, Chodkiewicz 30, Str., 85-064 Bydgoszcz, Poland. Electronic address: joanna.moraczewska@ukw.edu.pl.
authors.forename: Joanna
authors.initials: J
authors.lastname: Moraczewska
mesh_terms: D008841:Actin Cytoskeleton / Q000737:chemistry / Q000378:metabolism*; D000199:Actins / Q000378:metabolism; D019943:Amino Acid Substitution; D000818:Animals; D002384:Catalysis; D002645:Chickens; D004789:Enzyme Activation; D006801:Humans; D009119:Muscle Contraction / Q000235:genetics; D009135:Muscular Diseases / Q000235:genetics; D009218:Myosins / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D051381:Rats; D014335:Tropomyosin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014336:Troponin / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000199:Actins; D014335:Tropomyosin; D014336:Troponin; D009218:Myosins
keywords: Actin; Congenital myopathy; Contraction; Regulation; Tropomyosin; Troponin