title: Conserved Asp-137 imparts flexibility to tropomyosin and affects function.
abstract: Tropomyosin (Tm) is an alpha-helical coiled-coil that controls muscle contraction by sterically regulating the myosin-actin interaction. Tm moves between three states on F-actin as either a uniform or a non-uniform semi-flexible rod. Tm is stabilized by hydrophobic residues in the "a" and "d" positions of the heptad repeat. The highly conserved Asp-137 is unusual in that it introduces a negative charge on each chain in a position typically occupied by hydrophobic residues. The occurrence of two charged residues in the hydrophobic region is expected to destabilize the region and impart flexibility. To determine whether this region is unstable, we have substituted hydrophobic Leu for Asp-137 and studied changes in Tm susceptibility to limited proteolysis by trypsin and changes in regulation. We found that native and Tm controls that contain Asp-137 were readily cleaved at Arg-133 with t 1/2 of 5 min. In contrast, the Leu-137 mutant was not cleaved under the same conditions. Actin stabilized Tm, causing a 10-fold reduction in the rate of cleavage at Arg-133. The actin-myosin subfragment S1 ATPase activity was greater for the Leu mutant compared with controls in the absence of troponin and in the presence of troponin and Ca2+. We conclude that the highly conserved Asp-137 destabilizes the middle of Tm, resulting in a more flexible region that is important for the cooperative activation of the thin filament by myosin. We thus have shown a link between the dynamic properties of Tm and its function.
authors.affiliation: Cardiovascular Program, Boston Biomedical Research Institute, Watertown, Massachusetts 02472, USA.
authors.forename: John P
authors.initials: JP
authors.lastname: Sumida
authors.forename: Eleanor
authors.initials: E
authors.lastname: Wu
authors.forename: Sherwin S
authors.initials: SS
authors.lastname: Lehrer
mesh_terms: D000199:Actins / Q000737:chemistry / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000737:chemistry; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry*; D002645:Chickens; D017124:Conserved Sequence; D008954:Models, Biological; D009154:Mutation; D009218:Myosins / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D014335:Tropomyosin / Q000737:chemistry*; D014357:Trypsin / Q000737:chemistry
chemical_list: D000199:Actins; D011994:Recombinant Proteins; D014335:Tropomyosin; D001224:Aspartic Acid; D014357:Trypsin; D000251:Adenosine Triphosphatases; D009218:Myosins