The goal of the research is to understand the mechanism of thin filament regulation of contraction. The full activation of the thin filament by myosin, and Ca2+ binding to troponin C in thin filaments containing troponin (Tn), is cooperative (allosteric); tropomyosin (TM) is the agent for cooperativity. The alternatively expressed exons encoding the N- and C-terminal ends of TM are the major determinants of both universal and isoform-specific TM functions, consistent with extensive work showing that N- to C-terminal interactions of TM specific TM functions, consistent with extensive work showing that N-to C-terminal interactions of TM molecules along F-actin are essential for function. Troponin T binds to TM ends and modifies TM's function. Mutations in TM and Tn genes give rise to Familial Hypertrophic Cardiomyopathy and Nemaline Myopathy. At this time there is limited structural information about the critical functional domains of TM and TnT. Circular dichroism spectrometry and heteronuclear NMR, as well as other biochemical methods, will be used to investigate the structures of synthetic and recombinant N- and C-TM peptides, and the N-terminus of TnT, and the interactions between them. The new knowledge about the structure of the primary functional domains of TM and TnT that will come from the proposed work is a key step towards revealing a fundamental and significant property of the actin filament and cytoskeleton: how TM confers cooperativity to the thin filament and how information is communicated cooperatively along the thin filament. There are four specific aims. 1. To determine the structure of the non-muscle alpha-tropomyosin N- terminus encoded by exon 1b, and refinement of the structure of the exon 1a N-terminus, found in sarcomeric tropomyosins. 2. To determine the structure of the C-terminus of alpha/alpha- tropomyosin. Does it form a two-stranded coiled coil? 3. To determine the interaction and the structure of the overlap region between the N- and C- terminal domains of alpha/alpha-tropomyosin. 4. To determine the structure of the N-terminal, tropomyosin-binding, regulatory domain of human cardiac TnT, and the effect of FHC mutations on its structure and function.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM036326-12A1
Application #
6011704
Study Section
Physiological Chemistry Study Section (PC)
Program Officer
Lymn, Richard W
Project Start
1985-09-01
Project End
2003-06-30
Budget Start
1999-07-01
Budget End
2000-06-30
Support Year
12
Fiscal Year
1999
Total Cost
Indirect Cost
Name
University of Medicine & Dentistry of NJ
Department
Neurosciences
Type
Schools of Medicine
DUNS #
622146454
City
Piscataway
State
NJ
Country
United States
Zip Code
08854
Chang, Audrey N; Greenfield, Norma J; Singh, Abhishek et al. (2014) Structural and protein interaction effects of hypertrophic and dilated cardiomyopathic mutations in alpha-tropomyosin. Front Physiol 5:460
Oguchi, Yusuke; Ishizuka, Junji; Hitchcock-DeGregori, Sarah E et al. (2011) The role of tropomyosin domains in cooperative activation of the actin-myosin interaction. J Mol Biol 414:667-80
Hitchcock-DeGregori, Sarah E; Singh, Abhishek (2010) What makes tropomyosin an actin binding protein? A perspective. J Struct Biol 170:319-24
Greenfield, Norma J; Kotlyanskaya, Lucy; Hitchcock-DeGregori, Sarah E (2009) Structure of the N terminus of a nonmuscle alpha-tropomyosin in complex with the C terminus: implications for actin binding. Biochemistry 48:1272-83
Singh, Abhishek; Hitchcock-Degregori, Sarah E (2009) A peek into tropomyosin binding and unfolding on the actin filament. PLoS One 4:e6336
Hitchcock-DeGregori, Sarah E (2008) Tropomyosin: function follows structure. Adv Exp Med Biol 644:60-72
Kostyukova, Alla S; Hitchcock-Degregori, Sarah E; Greenfield, Norma J (2007) Molecular basis of tropomyosin binding to tropomodulin, an actin-capping protein. J Mol Biol 372:608-18
Wawro, Barbara; Greenfield, Norma J; Wear, Martin A et al. (2007) Tropomyosin regulates elongation by formin at the fast-growing end of the actin filament. Biochemistry 46:8146-55
Hitchcock-DeGregori, Sarah E; Greenfield, Norma J; Singh, Abhishek (2007) Tropomyosin: regulator of actin filaments. Adv Exp Med Biol 592:87-97
Greenfield, Norma J; Huang, Yuanpeng Janet; Swapna, G V T et al. (2006) Solution NMR structure of the junction between tropomyosin molecules: implications for actin binding and regulation. J Mol Biol 364:80-96

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