title: Mini-thin filaments regulated by troponin-tropomyosin.
abstract: Striated muscle thin filaments contain hundreds of actin monomers and scores of troponins and tropomyosins. To study the cooperative mechanism of thin filaments, "mini-thin filaments" were generated by isolating particles nearly matching the minimal structural repeat of thin filaments: a double helix of actin subunits with each strand approximately seven actins long and spanned by a troponin-tropomyosin complex. One end of the particles was capped by a gelsolin (segment 1-3)-TnT fusion protein (substituting for normal TnT), and the other end was capped by tropomodulin. EM showed that the particles were 46 +/- 9 nm long, with a knob-like mass attributable to gelsolin at one end. Average actin, tropomyosin, and gelsolin-troponin composition indicated one troponin-tropomyosin attached to each strand of the two-stranded actin filament. The minifilaments thus nearly represent single regulatory units of thin filaments. The myosin S1 MgATPase rate stimulated by the minifilaments was Ca2+-sensitive, indicating that single regulatory length particles are sufficient for regulation. Ca2+ bound cooperatively to cardiac TnC in conventional thin filaments but noncooperatively to cardiac TnC in minifilaments in the absence of myosin. This suggests that thin filament Ca2+-binding cooperativity reflects indirect troponin-troponin interactions along the long axis of conventional filaments, which do not occur in minifilaments. Despite noncooperative Ca2+ binding to minifilaments in the absence of myosin, Ca2+ cooperatively activated the myosin S1-particle ATPase rate. Two-stranded single regulatory units therefore may be sufficient for myosin-mediated Ca2+-binding cooperativity. Functional mini-thin filaments are well suited for biochemical and structural analysis of thin-filament regulation.
authors.affiliation: Department of Internal Medicine, University of Iowa, Iowa City, IA 52242, USA.
authors.forename: Huiyu
authors.initials: H
authors.lastname: Gong
authors.forename: Victoria
authors.initials: V
authors.lastname: Hatch
authors.forename: Laith
authors.initials: L
authors.lastname: Ali
authors.forename: William
authors.initials: W
authors.lastname: Lehman
authors.forename: Roger
authors.initials: R
authors.lastname: Craig
authors.forename: Larry S
authors.initials: LS
authors.lastname: Tobacman
mesh_terms: D008841:Actin Cytoskeleton / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure*; D000199:Actins; D000494:Allosteric Regulation; D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D018260:Gelsolin; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D018482:Muscle, Skeletal / Q000648:ultrastructure; D015879:Myosin Subfragments / Q000378:metabolism*; D010316:Particle Size; D011485:Protein Binding; D014335:Tropomyosin / Q000378:metabolism / Q000502:physiology*; D014336:Troponin / Q000378:metabolism / Q000502:physiology*
chemical_list: D000199:Actins; D018260:Gelsolin; D046911:Macromolecular Substances; D015879:Myosin Subfragments; D014335:Tropomyosin; D014336:Troponin; D017301:Ca(2+) Mg(2+)-ATPase; D002118:Calcium