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The nemaline myopathy and cap disease-associated Glu41Lys mutation in β-tropomyosin (TM) has been shown to alter contractile regulation. To study how the Glu41Lys mutation affects the position and flexibility of TM on the thin filament, we labelled recombinant wild type and mutant β-TMs with 5-IAF and F-actin with FITC-phalloidin, and incorporated them into ghost muscle fibres. The orientation and mobility of the probes were studied by polarized fluorimetry at different stages of the ATPase cycle. Multistep alterations in the position and flexibility of β-TM strands and actin monomers in the thin filaments were observed. In the absence of myosin heads the Glu41Lys mutation was found to shift tropomyosin strands towards the outward domain of actin, exposing few myosin-binding sites. The Glu41Lys mutation inhibited myosin-induced shift of TM strands towards the open position, markedly changing the flexural rigidity of both F-actin and TM strands and decreasing the amount of actin monomers capable of forming cross-bridges. This indicates that the number of the cross-bridges that are strongly bound during of the ATPase cycle is reduced. These structural changes in the thin filament are likely to underlie the observed decrease in Ca2+-sensitivity caused by this mutation, the alteration which plausibly initiates the disease. The work was supported by the Russian Fund for Fundamental Research (№ 11-04-00244a), the Programme of Presidium of RAS (theme № 7) and the Muscular Dystrophy Campaign.