Abstract

The broad long-term objective of this proposal is to elucidate the function of tropomyosin in the regulation of striated and smooth muscle contraction. This requires a detailed knowledge of its interactions with actin and troponin as modulated by myosin, Ca2+, and ATP. Fluorescence measurements of probes attached to cysteine groups of tropomyosin, will be used to monitor the cooperative change in state of tropomyosin associated with myosin subfragment 1 binding to the thin filament. Specifically, the measurements will be used to: show that the tropomyosin state is correlated with acto-myosin subfragment 1 ATPase activity, clarify the role of the two heads of myosin, determine the influence of the cardiac and skeletal troponin components on the tropomyosin state, verify that tropomyosin states, and determine the differences in behavior of smooth and striated muscle tropomyosins. Circular dichroism, fluorescence and chemical modification methods will be used to study the mechanism of assembly of the alpha and beta tropomyosin subunits (whose amino acid sequences slightly differ) into dimers (alphaalpha, alphabeta and betabeta). The conformational and regulatory properties of each dimer will be characterized by unfolding/assembly studies using tropomyosin from rabbit and frog striated muscle, chicken gizzard and bovine aorta smooth muscle, and mutated striated and smooth alphaalpha tropomyosin prepared by recombinant DNA techniques. The relationships between subunit amino acid will allow the characterization of normal genetic variation as well as alterations associated with genetic defects. Myosin rod (smooth and striated) unfolding/assembly studies will be continued to clarify conformational changes in specific regions of the rod associated with filament formation and activity. These tropomyosin and myosin conformational studies also provide basic information about folding intermediates and assembly mechanisms applicable to other proteins with similar coiled-coil structures.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL022461-20
Application #
2838895
Study Section
Biophysical Chemistry Study Section (BBCB)
Project Start
1978-04-01
Project End
2000-11-30
Budget Start
1998-12-01
Budget End
1999-11-30
Support Year
20
Fiscal Year
1999
Total Cost
Indirect Cost

  Institution

Name
Boston Biomedical Research Institute
Department
Type
DUNS #
058893371
City
Watertown
State
MA
Country
United States
Zip Code
02472

  Publications

Janco, Miro; Suphamungmee, Worawit; Li, Xiaochuan et al. (2013) Polymorphism in tropomyosin structure and function. J Muscle Res Cell Motil 34:177-87
Mudalige, Wasana A K A; Tao, Terence C; Lehrer, Sherwin S (2009) Ca2+-dependent photocrosslinking of tropomyosin residue 146 to residues 157-163 in the C-terminal domain of troponin I in reconstituted skeletal muscle thin filaments. J Mol Biol 389:575-83
Sumida, John P; Wu, Eleanor; Lehrer, Sherwin S (2008) Conserved Asp-137 imparts flexibility to tropomyosin and affects function. J Biol Chem 283:6728-34
Bacchiocchi, Corrado; Graceffa, Philip; Lehrer, Sherwin S (2004) Myosin-induced movement of alphaalpha, alphabeta, and betabeta smooth muscle tropomyosin on actin observed by multisite FRET. Biophys J 86:2295-307
Chen, Yaodong; Lehrer, Sherwin S (2004) Distances between tropomyosin sites across the muscle thin filament using luminescence resonance energy transfer: evidence for tropomyosin flexibility. Biochemistry 43:11491-9
Bacchiocchi, Corrado; Lehrer, Sherwin S (2002) Ca(2+)-induced movement of tropomyosin in skeletal muscle thin filaments observed by multi-site FRET. Biophys J 82:1524-36
Geeves, Michael A; Lehrer, Sherwin S (2002) Cooperativity in the Ca2+ regulation of muscle contraction. Results Probl Cell Differ 36:111-32
Maytum, R; Konrad, M; Lehrer, S S et al. (2001) Regulatory properties of tropomyosin effects of length, isoform, and N-terminal sequence. Biochemistry 40:7334-41
Suarez, M C; Lehrer, S S; Silva, J L (2001) Local heterogeneity in the pressure denaturation of the coiled-coil tropomyosin because of subdomain folding units. Biochemistry 40:1300-7
Zhou, X; Morris, E P; Lehrer, S S (2000) Binding of troponin I and the troponin I-troponin C complex to actin-tropomyosin. Dissociation by myosin subfragment 1. Biochemistry 39:1128-32

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