title: Different positions of tropomyosin isoforms on actin filament are determined by specific sequences of end-to-end overlaps.
abstract: Tropomyosins are dimeric rod-like proteins which polymerize along actin filaments and regulate interactions with other actin-binding proteins. Homologous sequences responsible for the binding of tropomyosin to consecutive actin monomers repeat along tropomyosin and are called actin-binding periods. In this work, the localization of tropomyosin isoforms on actin alone and on actin–myosin complex was evaluated by measuring Förster resonance energy transfer (FRET) distances between a donor (AEDANS) attached to either the N-terminal actin-binding period 1 or to the central actin-binding period 5 and an acceptor (DABMI) bound to actin's Cys374. The recombinant -tropomyosin isoforms–TM2, TM5a, and TM1b9a, used in this study, had various amino acid sequences of the N- and C-termini forming the end-to-end overlap. Although the sequences of actin-binding period 5 of the three isoforms were identical, the donor–acceptor distances calculated for each isoform varied between 38.6 and 41.5 Å. Differences in FRET distances between the three tropomyosin isoforms labeled in actin-binding period 1 varied between 34.8 and 40.2 Å. Rigor binding of myosin heads to actin increased all measured distances. The degree and cooperativity of myosin-induced shift was different for each of the isoforms and actin-binding periods. The structural differences correlate with cooperative regulation of actin-activated S1 ATPase by the three tropomyosins. The results indicate that amino acid sequences of the end-to-end overlap determine specific orientation of tropomyosin isoform on actin. This can be important for steric and cooperative regulation of the actin filament and determine functional specificity of multiple tropomyosin isoforms present in eucaryotic cells.
authors.affiliation: Department of Biochemistry and Cell Biology, Kazimierz Wielki University, Institute of Experimental Biology, Bydgoszcz, Poland.
authors.forename: Małgorzata
authors.initials: M
authors.lastname: Sliwińska
authors.forename: Magdalena
authors.initials: M
authors.lastname: Zukowska
authors.forename: Danuta
authors.initials: D
authors.lastname: Borys
authors.forename: Joanna
authors.initials: J
authors.lastname: Moraczewska
mesh_terms: D000199:Actins / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D016880:Anisotropy; D031541:Fluorescence Resonance Energy Transfer; D009154:Mutation; D009218:Myosins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D014335:Tropomyosin / Q000235:genetics / Q000378:metabolism*
chemical_list: D000199:Actins; D014335:Tropomyosin; D009218:Myosins