title: Cardiomyopathic tropomyosin mutations that increase thin filament Ca2+ sensitivity and tropomyosin N-domain flexibility.
abstract: The relationship between tropomyosin thermal stability and thin filament activation was explored using two N-domain mutants of alpha-striated muscle tropomyosin, A63V and K70T, each previously implicated in familial hypertrophic cardiomyopathy. Both mutations had prominent effects on tropomyosin thermal stability as monitored by circular dichroism. Wild type tropomyosin unfolded in two transitions, separated by 10 degrees C. The A63V and K70T mutations decreased the melting temperature of the more stable of these transitions by 4 and 10 degrees C, respectively, indicating destabilization of the N-domain in both cases. Global analysis of all three proteins indicated that the tropomyosin N-domain and C-domain fold with a cooperative free energy of 1.0-1.5 kcal/mol. The two mutations increased the apparent affinity of the regulatory Ca2+ binding sites of thin filament in two settings: Ca2+-dependent sliding speed of unloaded thin filaments in vitro (at both pH 7.4 and 6.3), and Ca2+ activation of the thin filament-myosin S1 ATPase rate. Neither mutation had more than small effects on the maximal ATPase rate in the presence of saturating Ca2+ or on the maximal sliding speed. Despite the increased tropomyosin flexibility implied by destabilization of the N-domain, neither the cooperativity of thin filament activation by Ca2+ nor the cooperative binding of myosin S1-ADP to the thin filament was altered by the mutations. The combined results suggest that a more dynamic tropomyosin N-domain influences interactions with actin and/or troponin that modulate Ca2+ sensitivity, but has an unexpectedly small effect on cooperative changes in tropomyosin position on actin.
authors.affiliation: Departments of Internal Medicine and Biochemistry, University of Iowa, Iowa City, IA 52242, USA.
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Heller
authors.forename: Mahta
authors.initials: M
authors.lastname: Nili
authors.forename: Earl
authors.initials: E
authors.lastname: Homsher
authors.forename: Larry S
authors.initials: LS
authors.lastname: Tobacman
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D009202:Cardiomyopathies / Q000235:genetics*; D002417:Cattle; D007700:Kinetics; D009154:Mutation; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D011817:Rabbits; D051381:Rats; D013696:Temperature; D013816:Thermodynamics; D014335:Tropomyosin / Q000737:chemistry / Q000235:genetics*
chemical_list: D014335:Tropomyosin; D000251:Adenosine Triphosphatases; D002118:Calcium