Aberrant movement of b-tropomyosin associated with congenital myopathy causes defective response of myosin heads and actin during the ATPase cycle Yurii S. Borovikov a,⇑ , Stanislava V. Avrova a , Nikita A. Rysev a , Vladimir V. Sirenko a , Armen O. Simonyan a,b , Aleksey A. Chernev a,b , Olga E. Karpicheva a , Adam Piers c , Charles S. Redwood c a Institute of Cytology, Russian Academy of Sciences, 4 Tikhoretsky Avenue, St. Petersburg 194064, Russia b Saint Petersburg State University, Universitetskaya nab., 7-9, Saint Petersburg 199034, Russia c University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU, United Kingdom article info Article history: Received 13 January 2015 and in revised form 22 April 2015 Available online 13 May 2015 Keywords: Tropomyosin F-actin Myosin Troponin Ghost muscle fibers Congenital myopathies Fluorescence polarization abstract We have investigated the effect of the E41K, R91G, and E139del b-tropomyosin (TM) mutations that cause congenital myopathy on the position of TM and orientation of actin monomers and myosin heads at different mimicked stages of the ATPase cycle in troponin-free ghost muscle fibers by polarized fluo- rimetry. A multi-step shifting of wild-type TM to the filament center accompanied by an increase in the amount of switched on actin monomers and the strongly bound myosin heads was observed during the ATPase cycle. The R91G mutation shifts TM further towards the inner and outer domains of actin at the strong- and weak-binding stages, respectively. The E139del mutation retains TM near the inner domains, while the E41K mutation captures it near the outer domains. The E41K and R91G mutations can induce the strong binding of myosin heads to actin, when TM is located near the outer domains. The E139del mutation inhibits the amount of strongly bound myosin heads throughout the ATPase cycle. Ó 2015 Elsevier Inc. All rights reserved. Introduction Tropomyosin is a two-chained a-helical coiled-coil protein. Its rod-like molecules associate head-to-tail to form continuous strands that run along the entire length of F-actin. Amino acid sequence of the TM 1 molecule reflects the presence of 7 pseudo-repeats (about 40 amino acids each) that has been postu- lated to relate to the binding sites for the 7 actin monomers along the length of a TM molecule, conforming to the F-actin helix (for reviews, see [1,2]). TM’s role is best established in striated muscle where it cooperatively regulates contraction in response to Ca 2+ binding to troponin. Conformational changes in troponin induced by the binding of Ca 2+ act as a cooperative switch of the actin-tropomyosin interface between the relaxed and active states [3–5]. It is believed that in the absence of Ca 2+ troponin constrains TM in a position on the outer domain of actin, which inhibits the strong binding of myosin cross-bridges to actin (‘‘blocked position’’) and, consequently, the ATPase and muscle contraction. When Ca 2+ binds to troponin, it undergoes conformational changes and allows TM to rotate by 25° around the F-actin axis to move towards the inner domain of actin, thus exposing most of the myosin-binding sites [6,7]. However, TM still covers a part of the myosin-binding site (‘‘closed position’’). Only when myosin head attaches to the actin fil- ament, TM undergoes a 23-Å shift up and across with a 31° rotation around the F-actin axis [8] to move further to the inner domain of actin. As a result, TM takes a position near the inner domain of actin (‘‘open position’’) and fully exposes the myosin-binding site on actin [6,9]. These three structural states are in rapid equilibrium with each other [10], so that in each condition there is a distribution of states [11]. A significant role of conformational changes in actin in regula- tion of muscle contraction by TM was revealed in many works ([12–14] for reviews see [15]). Biochemical studies have shown that monomers in pure F-actin can exist in two states: the so-called ‘‘switched on’’ and ‘‘switched off’’. Unlike the switched off monomers, the switched on monomers are able to activate myosin ATPase, i.e. actin monomers may be in the so-called http://dx.doi.org/10.1016/j.abb.2015.05.002 0003-9861/Ó 2015 Elsevier Inc. All rights reserved. ⇑ Corresponding author. Fax: +7 8122970431. E-mail address: borovikov@incras.ru (Y.S. Borovikov). 1 Abbreviations used: TM, tropomyosin; WTTM, wild-type tropomyosin; S1, myosin subfragment-1; FITC-phalloidin, phalloidin-fluorescein isothiocyanate; 1,5-IAEDANS, N-(iodoacetaminoethyl)-1-naphthyl-amine-5-sulfonic acid; 5-IAF, 5-iodoacetamidofluorescein; DTT, dithiothreitol; NEM-S1, myosin subfragment-1 modified with N-ethylmaleimide; pPDM-S1, myosin subfragment-1 modified with N,N 0 -(1,2-phenylene)dimaleimide; AMP-PNP, adenosine 5 0 -(b,c-imido)triphosphate tetralithium salt hydrate. Archives of Biochemistry and Biophysics 577–578 (2015) 11–23 Contents lists available at ScienceDirect Archives of Biochemistry and Biophysics journal homepage: www.elsevier.com/locate/yabbi