The objective of this project is to determine structural interactions between cardiac troponin proteins responsible for regulation of heart muscle contraction. The central hypothesis is that a detailed structural understanding of the interactions between troponin proteins is essential to a molecular understanding of muscle contraction. The cardiac isoforms of both troponin C and troponin I are unique. The approach involves solution structure determination using heteronuclear multidimensional NMR in combination with a number of biochemical and biophysical techniques. Due to physical limitations of the troponin IC complex, we have developed a modular approach for studying this system based on the wealth of biochemical information available on the troponin complex.
The Specific Aim 1 is to determine solution structures for the Ca2+ - and Mg2+ - bound forms of cTnC(81-161)/cTnI(33-80). Secondary and tertiary structures of each protein in the complex will be determined by heteronuclear multidimensional NMR in combination with 15N.13C isotope labeling. During the contraction cycle, the two C- terminal Ca2+/Mg2+ sites alternate between the Ca2+-bound and Mg2+-bound forms. Conformational alterations induced by going between these two forms are unknown.
In Specific Aim 2 the structural effects of the cardiac specific NH2- terminus containing the PKA phosphorylation site will be identified. PKA phosphorylation is known to induce a new activity in the complex. The structural mechanism by which this occurs will be determined through NMR studies on the phosphorylated state.
The Specific Aim 3 is to determine the solution structures of apo- and Ca2+-saturated cTnC(1-86) by NMR. The isolated N-terminus domain of TnC has been shown to be a good model for the regulatory domain of TnC. Comparison of these two structures provides insight into the Ca2+ induced transition which initiates muscle contraction. The Ca2+ - saturated complex of cTC(1-90)/cTnI(86-211) will be determined in Specific Aim 4. This study is motivated by the need to understand Ca2+ dependent protein-protein interactions which initiate muscle contraction. Together, these studies will provide high resolution solution structures of the Ca2+/Mg2+ - dependent and Ca2+ - specific interaction domains within the cardiac troponin IC complex.
The Specific Aim 5 is to obtain selective structural information on the intact cTnIC complex. Since the molecular mass of TnIC is at the upper limit for structure determination by NMR, our strategy will be to obtain selective information using a combination of 15N/13C isotope labeling and/or deuteration. Assignments and 'models' developed in Specific Aims 1-4 will be used to facilitate assignment and secondary structure determination of the intact complex. Special emphasis will be placed on the 31 residue linker in cTnC and the central spiral including the inhibitory region of cTnI.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR044324-03
Application #
2837559
Study Section
Biophysical Chemistry Study Section (BBCB)
Program Officer
Lymn, Richard W
Project Start
1996-12-01
Project End
2000-11-30
Budget Start
1998-12-01
Budget End
1999-11-30
Support Year
3
Fiscal Year
1999
Total Cost
Indirect Cost
Name
University of Cincinnati
Department
Genetics
Type
Schools of Medicine
DUNS #
City
Cincinnati
State
OH
Country
United States
Zip Code
45221
Finley, Natosha L; Howarth, Jack W; Rosevear, Paul R (2004) Structure of the Mg2+-loaded C-lobe of cardiac troponin C bound to the N-domain of cardiac troponin I: comparison with the Ca2+-loaded structure. Biochemistry 43:11371-9
Finley, Natosha L; Rosevear, Paul R (2004) Introduction of negative charge mimicking protein kinase C phosphorylation of cardiac troponin I. Effects on cardiac troponin C. J Biol Chem 279:54833-40
Heller, William T; Finley, Natosha L; Dong, Wen-Ji et al. (2003) Small-angle neutron scattering with contrast variation reveals spatial relationships between the three subunits in the ternary cardiac troponin complex and the effects of troponin I phosphorylation. Biochemistry 42:7790-800
Dvoretsky, Alex; Gaponenko, Vadim; Rosevear, Paul R (2002) Derivation of structural restraints using a thiol-reactive chelator. FEBS Lett 528:189-92
Heller, William T; Abusamhadneh, Ekram; Finley, Natosha et al. (2002) The solution structure of a cardiac troponin C-troponin I-troponin T complex shows a somewhat compact troponin C interacting with an extended troponin I-troponin T component. Biochemistry 41:15654-63
Dvoretsky, Alex; Abusamhadneh, Ekram M; Howarth, Jack W et al. (2002) Solution structure of calcium-saturated cardiac troponin C bound to cardiac troponin I. J Biol Chem 277:38565-70
Sorsa, T; Heikkinen, S; Abbott, M B et al. (2001) Binding of levosimendan, a calcium sensitizer, to cardiac troponin C. J Biol Chem 276:9337-43
Abbott, M B; Dong, W J; Dvoretsky, A et al. (2001) Modulation of cardiac troponin C-cardiac troponin I regulatory interactions by the amino-terminus of cardiac troponin I. Biochemistry 40:5992-6001
Abusamhadneh, E; Abbott, M B; Dvoretsky, A et al. (2001) Interaction of bepridil with the cardiac troponin C/troponin I complex. FEBS Lett 506:51-4
Gaponenko, V; Dvoretsky, A; Walsby, C et al. (2000) Calculation of z-coordinates and orientational restraints using a metal binding tag. Biochemistry 39:15217-24

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