The primary cause of muscle disfunction associated with substitutions E240K and R244G in tropomyosin is aberrant behavior of tropomyosin and response of actin and myosin during ATPase cycle

Using the polarized photometry technique we have studied the effects of two amino acid replacements, E240K and R244G, in tropomyosin (Tpm1.1) on the position of Tpm1.1 on troponin-free actin filaments and the spatial arrangement of actin monomers and myosin heads at various mimicked stages of the ATPase cycle in the ghost muscle fibres. E240 and R244 are located in the C-terminal, seventh actin-binding period, in f and b positions of the coiled-coil heptapeptide repeat. Actin, Tpm1.1, and myosin subfragment-1 (S1) were fluorescently labeled: 1.5-IAEDANS was attached to actin and S1, 5-IAF was bound to Tpm1.1. The labeled proteins were incorporated in the ghost muscle fibres and changes in polarized fluorescence during the ATPase cycle have been measured. It was found that during the ATPase cycle both mutant tropomyosins occupied a position close to the inner domain of actin. The relative amount of the myosin heads in the strongly-bound conformations and of the switched on actin monomers increased at mimicking different stages of the ATPase cycle. This might be one of the reasons for muscle dysfunction in congenital fibre type disproportion caused by the substitutions E240K and R244G in tropomyosin. [Display omitted] •We used polarized fluorimetry to assess the conformation changes in S1, tropomyosin and actin during the ATPase cycle.•Myosin SH1 helix, tropomyosin and actin subdomain-1 movements were monitored.•E240K and R244G Tpm1.1 mutants were compared with the wild type Tpm1.1.•E240K and R244G mutations shift the tropomyosin strands towards the inner domain of actin.•The Tpm1.1 mutants in muscle fiber increase the amount of the myosin heads in the strong-binding conformation.