title: Effects of the amino-terminal regions of tropomyosin and troponin T on thin filament assembly.
abstract: Bacterially expressed alpha-tropomyosin lacks the amino-terminal acetylation present in muscle tropomyosin and binds poorly to actin (Hitchcock-DeGregori, S. E., and Heald, R. W. (1987) J. Biol. Chem. 262, 9730-9735). Using a linear lattice model, we determined the affinity (Ko) of unacetylated tropomyosin or troponin-unacetylated tropomyosin for an isolated site on the actin filament and the fold increase in affinity (y) when binding is to an adjacent site. The absence of tropomyosin acetylation decreased Ko 2 orders of magnitude in the absence of troponin. Tropomyosin acetylation also enhanced troponin-tropomyosin binding to actin, not by increasing cooperativity (y), but rather by increasing Ko. These results suggest that the amino-terminal region of tropomyosin is a crucial actin binding site. Troponin promoted unacetylated tropomyosin binding to actin, increasing Ko more than 1,000-fold. Troponin70-259, which lacks the troponin T peptide (1-69) spanning the overlap between adjacent tropomyosins, behaved similarly to intact troponin. Cooperative interactions between adjacent troponin-tropomyosin complexes remained strong despite the use of a nonpolymerizable tropomyosin and a troponin unable to bridge neighboring tropomyosins physically. The Ko for troponin70-259-unacetylated tropomyosin was 500-fold greater than for troponin159-259-unacetylated tropomyosin, indicating that troponin T residues 70-158 are critical for anchoring troponin-tropomyosin to F-actin. The mechanism of cooperative thin filament assembly is discussed.
authors.affiliation: Department of Internal Medicine, College of Medicine, University of Iowa, Iowa City 52242.
authors.forename: K A
authors.initials: KA
authors.lastname: Willadsen
authors.forename: C A
authors.initials: CA
authors.lastname: Butters
authors.forename: L E
authors.initials: LE
authors.lastname: Hill
authors.forename: L S
authors.initials: LS
authors.lastname: Tobacman
mesh_terms: D000107:Acetylation; D000199:Actins / Q000302:isolation & purification / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008433:Mathematics; D009132:Muscles / Q000378:metabolism; D015879:Myosin Subfragments / Q000302:isolation & purification / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D014335:Tropomyosin / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014336:Troponin / Q000302:isolation & purification / Q000378:metabolism*; D020107:Troponin T
chemical_list: D000199:Actins; D015879:Myosin Subfragments; D011994:Recombinant Proteins; D014335:Tropomyosin; D014336:Troponin; D020107:Troponin T; D004247:DNA