The long term goal of this project is to elucidate the molecular basis of contraction and regulation in striated muscle. This field is now in a revolutionary phase due to advances in genetic, crystallographic, and cryo-electron microscopic techniques. Our approach is to use cryo-electron microscopy to define the molecular structures of the actin and myosin filaments and to capture the dynamic molecular events that generate and regulate contraction. By combining our observations with the atomic structures of actin and the myosin head, we are approaching a near-atomic description of contractile events.
Our Specific Aims are: (1) To define the conformation, disposition and interactions of the myosin heads (crossbridges) that characterize the relaxed state of myosin filaments. (2) To determine, on the millisecond time-scale, the structural changes that occur in the crossbridges on the myosin filaments when they are activated by Ca2+ binding or by light chain phosphorylation. (3) To define the position of tropomyosin in the actin filaments of relaxed muscle and the changes in position that characterize the activated state, when troponin binds Ca2+. (4) To capture cross bridge motions during the crossbridge cycle in vitro. The use of cryo-electron microscopy is key to this proposal. Specimens are rapidly frozen, which preserves native filament structure and arrests transient molecular conformations, and are then observed in the unstained, frozen-hydrated state. Techniques will be used to generate rapid (millisecond time-scale) transitions in Ca2+ and ATP levels, allowing dynamic events of contraction to be captured. Three-dimensional structures of filaments will be computed by Fourier-based three-dimensional reconstruction methods, and molecular fitting will be used to relate our results to atomic structures. Preliminary data show that all of these goals are feasible. This project will provide data crucial to our understanding of the molecular mechanism of contraction and its regulation. It will help to illuminate general mechanisms of actin-myosin based cell motility in nonmuscle cells. And it will deepen our understanding of the structure of healthy muscle, which is essential to an understanding of structural defects that occur in diseased states.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR034711-11
Application #
2683271
Study Section
Molecular Cytology Study Section (CTY)
Project Start
1984-12-01
Project End
2000-03-31
Budget Start
1998-04-01
Budget End
1999-03-31
Support Year
11
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of Massachusetts Medical School Worcester
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
660735098
City
Worcester
State
MA
Country
United States
Zip Code
01655
Lee, Kyoung Hwan; Sulbarán, Guidenn; Yang, Shixin et al. (2018) Interacting-heads motif has been conserved as a mechanism of myosin II inhibition since before the origin of animals. Proc Natl Acad Sci U S A 115:E1991-E2000
Mun, Ji Young; Kensler, Robert W; Harris, Samantha P et al. (2016) The cMyBP-C HCM variant L348P enhances thin filament activation through an increased shift in tropomyosin position. J Mol Cell Cardiol 91:141-7
Previs, Michael J; Mun, Ji Young; Michalek, Arthur J et al. (2016) Phosphorylation and calcium antagonistically tune myosin-binding protein C's structure and function. Proc Natl Acad Sci U S A 113:3239-44
Yang, Shixin; Woodhead, John L; Zhao, Fa-Qing et al. (2016) An approach to improve the resolution of helical filaments with a large axial rise and flexible subunits. J Struct Biol 193:45-54
Previs, Michael J; Prosser, Benjamin L; Mun, Ji Young et al. (2015) Myosin-binding protein C corrects an intrinsic inhomogeneity in cardiac excitation-contraction coupling. Sci Adv 1:
Sulbarán, Guidenn; Alamo, Lorenzo; Pinto, Antonio et al. (2015) An invertebrate smooth muscle with striated muscle myosin filaments. Proc Natl Acad Sci U S A 112:E5660-8
Lee, Kyounghwan; Harris, Samantha P; Sadayappan, Sakthivel et al. (2015) Orientation of myosin binding protein C in the cardiac muscle sarcomere determined by domain-specific immuno-EM. J Mol Biol 427:274-86
Kirk, Jonathan A; Chakir, Khalid; Lee, Kyoung Hwan et al. (2015) Pacemaker-induced transient asynchrony suppresses heart failure progression. Sci Transl Med 7:319ra207
Mun, Ji Young; Previs, Michael J; Yu, Hope Y et al. (2014) Myosin-binding protein C displaces tropomyosin to activate cardiac thin filaments and governs their speed by an independent mechanism. Proc Natl Acad Sci U S A 111:2170-5
Craig, Roger; Lee, Kyoung Hwan; Mun, Ji Young et al. (2014) Structure, sarcomeric organization, and thin filament binding of cardiac myosin-binding protein-C. Pflugers Arch 466:425-31

Showing the most recent 10 out of 88 publications