Thin filament-linked actin-binding proteins control both actomyosin-based muscle contraction and cytoskeletal formation. In order to understand the normal control of muscle contraction, it is crucial to characterize the structural interactions of thin filament regulatory proteins that influence actin-myosin association. Using structural analysis, we will examine the architecture of cardiac and skeletal muscle thin filaments at a fundamental level and determine the changing interactions of thin filament-linked proteins that regulate muscle activity. Our principal objective therefore is to solve the atomic structure of the entire thin filament in relaxed and in active muscle. We use state-of-the-art electron microscopy and electron tomography. coupled with image analysis, 3D reconstruction (3D-EM) and computational chemistry to establish the macromolecular structure of actin-binding proteins on thin filament actin and thus demarcate molecular contacts of binding proteins with actin at near atomic resolution. Using these techniques: (1) We aim to determine the structural basis of troponin-tropomyosin regulation of cardiac and skeletal muscle activity by analyzing interactions of tropomyosin and troponin on thin filaments, which are governed by Ca2+-binding to troponin and myosin-crossbridge binding on actin. To accomplish this goal, (a) we will describe the complete atomic structure of tropomyosin and troponin-tropomyosin on thin filaments by generating single particle and electron tomographic reconstructions at increasingly high resolution and by fitting atomic resolution crystal structures of actin and regulatory proteins into the reconstruction volumes; (b) we will further refine these atomic structures using computational tools to maximize chemical interactions between regulatory proteins and actin; (c) we will use Molecular Dynamics protocols to assess regulatory protein dynamics and likely transitions between regulatory states. (2) Using this same approach, we will test the hypothesis that mutant cardiac troponin and tropomyosin perturb muscle regulation by causing imbalanced protein interactions that alter the regulatory state of thin filaments, and we will determine the underlying structural reasons for such perturbations. (3) We will test the hypothesis that the short molecular overlap domain between adjacent tropomyosins, needed for end-to-end tropomyosin linkage, is vital for tropomyosin strand formation on filaments, and again assess the impact of mutants on this structure. (4) We will complete efforts to test the hypothesis that Myosin-Binding Protein C forms regular and periodic links to thin filaments, which are likely to modulate striated contractile activity.

Public Health Relevance

Studies on tropomyosin- and troponin-regulated filaments, with particular attention devoted to normal and mutant proteins, will lead to an elucidation of the molecular regulatory mechanisms governing cardiac muscle contraction. This work is essential for unraveling cardiomyopathic disease processes and revealing key controls for vascular tone and pulmonary airway resistance, determinants in, for example, hypertension and asthma. Actin filaments and their associated proteins are major participants in diverse cellular systems having varied function, thus underscoring the broad significance of the proposed work.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37HL036153-27
Application #
9184575
Study Section
Special Emphasis Panel (NSS)
Program Officer
Gao, Yunling
Project Start
1986-09-30
Project End
2017-12-31
Budget Start
2016-12-01
Budget End
2017-12-31
Support Year
27
Fiscal Year
2017
Total Cost
Indirect Cost
Name
Boston University
Department
Physiology
Type
Schools of Medicine
DUNS #
604483045
City
Boston
State
MA
Country
United States
Zip Code
02118
Farman, Gerrie P; Rynkiewicz, Michael J; Orzechowski, Marek et al. (2018) HCM and DCM cardiomyopathy-linked ?-tropomyosin mutations influence off-state stability and crossbridge interaction on thin filaments. Arch Biochem Biophys 647:84-92
Rynkiewicz, Michael J; Prum, Thavanareth; Hollenberg, Stephen et al. (2017) Tropomyosin Must Interact Weakly with Actin to Effectively Regulate Thin Filament Function. Biophys J 113:2444-2451
Lehman, William (2017) Switching Muscles On and Off in Steps: The McKillop-Geeves Three-State Model of Muscle Regulation. Biophys J 112:2459-2466
Moore, Jeffrey R; Campbell, Stuart G; Lehman, William (2016) Structural determinants of muscle thin filament cooperativity. Arch Biochem Biophys 594:8-17
Lehman, William (2016) Thin Filament Structure and the Steric Blocking Model. Compr Physiol 6:1043-69
Rynkiewicz, Michael J; Fischer, Stefan; Lehman, William (2016) The propensity for tropomyosin twisting in the presence and absence of F-actin. Arch Biochem Biophys 609:51-58
Sewanan, Lorenzo R; Moore, Jeffrey R; Lehman, William et al. (2016) Predicting Effects of Tropomyosin Mutations on Cardiac Muscle Contraction through Myofilament Modeling. Front Physiol 7:473
Fischer, Stefan; Rynkiewicz, Michael J; Moore, Jeffrey R et al. (2016) Tropomyosin diffusion over actin subunits facilitates thin filament assembly. Struct Dyn 3:012002
Schmidt, William M; Lehman, William; Moore, Jeffrey R (2015) Direct observation of tropomyosin binding to actin filaments. Cytoskeleton (Hoboken) 72:292-303
Alamo, Lorenzo; Li, Xiaochuan Edward; Espinoza-Fonseca, L Michel et al. (2015) Tarantula myosin free head regulatory light chain phosphorylation stiffens N-terminal extension, releasing it and blocking its docking back. Mol Biosyst 11:2180-9

Showing the most recent 10 out of 39 publications