ИСТИНА

Previous analyses showed that mutations in TPM1 gene, which resulted in two substitutions: Arg167His and Lys168Glu, in a-tropomyosin (Tpm1.1), caused severe dysfunctions in Ca-dependent activation of actin-myosin ATPase. The goal of the present study was to investigate the effects of the mutations on the orientation of troponin-tropomyosin complex on actin filament at different stages of the actomyosin ATPase cycle. The recombinant wild-type and mutant Tpm1.1, myosin subfragment-1 (S1) and F-actin were labeled respectively with 5-IAF, 1,5-IAEDANS and FITC-phalloidin. The fluorescently labeled proteins were incorporated into ghost muscle fibres and used for the analysis of polarized fluorescence. The data allowed us to estimate the extend of Ca2+-induced shift in the position of the wild-type and mutant Tpm1.1 on the filament, the fraction of actin monomers switched into the ‘on’ state and the fraction of myosin S1 bound in rigor. At low Ca2+ the substitution Arg167Gly shifted tropomyosin strands towards inner domain of actin and the fraction of the actin subunits in the ‘on’ state was increased as compared to the wildtype tropomyosin. In the presence of Ca2+ this mutation caused a shift of tropomyosin towards outer domain of actin and did not change the fraction of the actin subunits in ‘on’ state. This tendency was maintained throughout the ATPase cycle, resulting in inhibition of strong-binding state of actomyosin. This indicates that the mutations Arg167Gly in Tpm1.1 affected troponin’s ability to switch off actin monomers at low Ca2+ and to activate the crossbridges at high Ca2+. In contrast, tropomyosin carrying the substitution Lys168Glu was shifted towards the outer actin domain at both low and high Ca2+. The fraction of the actin subunits in ‘on’ state did not change at high Ca2+, while the amount of the myosin heads strongly bound to F-actin decreased at high Ca2+, but increased at low Ca2+, showing that troponin lost its ability to fully activate the cross-bridges at high Ca2+ and to deactivate them at low Ca2+. The data indicate that both mutations disrupt the regulatory functions of troponin. Supported by the Russian Foundation for Basic Research (Grants No. 14-04-00454a, 14-04-31527a).