title: Three-dimensional organization of troponin on cardiac muscle thin filaments in the relaxed state.
abstract: Muscle contraction is regulated by troponin-tropomyosin, which blocks and unblocks myosin binding sites on actin. To elucidate this regulatory mechanism, the three-dimensional organization of troponin and tropomyosin on the thin filament must be determined. Although tropomyosin is well defined in electron microscopy helical reconstructions of thin filaments, troponin density is mostly lost. Here, we determined troponin organization on native relaxed cardiac muscle thin filaments by applying single particle reconstruction procedures to negatively stained specimens. Multiple reference models led to the same final structure, indicating absence of model bias in the procedure. The new reconstructions clearly showed F-actin, tropomyosin, and troponin densities. At the 25 Å resolution achieved, troponin was considerably better defined than in previous reconstructions. The troponin density closely resembled the shape of troponin crystallographic structures, facilitating detailed interpretation of the electron microscopy density map. The orientation of troponin-T and the troponin core domain established troponin polarity. Density attributable to the troponin-I mobile regulatory domain was positioned where it could hold tropomyosin in its blocking position on actin, thus suggesting the underlying structural basis of thin filament regulation. Our previous understanding of thin filament regulation had been limited to known movements of tropomyosin that sterically block and unblock myosin binding sites on actin. We now show how troponin, the Ca(2+) sensor, may control these movements, ultimately determining whether muscle contracts or relaxes.
authors.affiliation: Department of Cell and Developmental Biology, University of Massachusetts Medical School, Worcester, Massachusetts.
authors.forename: Shixin
authors.initials: S
authors.lastname: Yang
authors.affiliation: Faculty of Biology and Geology, Babeş-Bolyai University, Cluj-Napoca, Romania.
authors.forename: Lucian
authors.initials: L
authors.lastname: Barbu-Tudoran
authors.affiliation: Department of Physiology and Biophysics, Boston University School of Medicine, Boston, Massachusetts.
authors.forename: Marek
authors.initials: M
authors.lastname: Orzechowski
authors.affiliation: Department of Cell and Developmental Biology, University of Massachusetts Medical School, Worcester, Massachusetts.
authors.forename: Roger
authors.initials: R
authors.lastname: Craig
authors.affiliation: School of Molecular and Cellular Biology, University of Leeds, Leeds, United Kingdom.
authors.forename: John
authors.initials: J
authors.lastname: Trinick
authors.affiliation: Department of Physiological Sciences, Eastern Virginia Medical School, Norfolk, Virginia.
authors.forename: Howard
authors.initials: H
authors.lastname: White
authors.affiliation: Department of Physiology and Biophysics, Boston University School of Medicine, Boston, Massachusetts. Electronic address: wlehman@bu.edu.
authors.forename: William
authors.initials: W
authors.lastname: Lehman
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003599:Cytoskeleton / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure*; D008969:Molecular Sequence Data; D032383:Myocytes, Cardiac / Q000737:chemistry / Q000648:ultrastructure*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013552:Swine; D014335:Tropomyosin / Q000737:chemistry / Q000378:metabolism; D014336:Troponin / Q000737:chemistry* / Q000378:metabolism
chemical_list: D014335:Tropomyosin; D014336:Troponin