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.
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