| At the heart of muscle regulation mechanism is troponin C (TnC), the Ca2+-binding component of the troponin (Tn) complex. In Chapter II, interactions of EMD 57033, a Ca2+-sensitizer, with the C-terminal domain of cardiac TnC (cCTnC) is investigated. The structure of cCTnC complexed to EMD 57033 showed that the drug binds to the hydrophobic groove on the domain and the orientation of the binding is such that the methyl group on the stereo centre of the drug makes contacts with residues in the core of the domain. The enantiomer of EMD 57033 was shown to interact differently with cCTnC than EMD 57033.;In Chapter III, the interaction of bepridil, another Ca2+-sensitizer, with the N-terminal domain of cTnC (cNTnC) is studied. Bepridil is shown to weaken cTnC binding to the switch region of cTnI (cSp). The tertiary structure of the cNTnC•cSp•bepridil complex revealed that both bepridil and the N-terminal of cSp bind to the same hydrophobic region on cNTnC such bepridil displaces the N-terminal of cSp from its binding site, weakening the affinity of cSp for cTnC.;In Chapter IV, the effect of phosphorylation at residue S149 of cTnI on cTnI-cTnC interactions is investigated. The phosphorylation of S149 weakens the affinity of cSp for cTnC owing to the disruption of important cTnI-cTnC hydrophobic contacts. However, S149-phosphorylated cTnI showed a significant increase in its affinity for cTnC in the presence of the FHC mutation R161W.;Chapters V to VII are devoted to the development of an in situ 19FNMR method that can be applied to myofibrils containing 19F-labelled cTnC. Chapter V explains the theoretical requirements needed to make the experiment feasible. It also examines the effect of the distribution of Tn on thin filament on the expected result of the experiment. Chapter VI focuses on methods for expressing 19F-labelled proteins. In particular, it examines the correlation between the type of fluorinated amino acid and the degree of perturbation to the TnC structure. In Chapter VII, the structures of two fluorinated Phe-to-Trp mutants of cTnC are closely examined. The mutations themselves are found to produce no significant perturbation to the structure of cTnC. The mutants have a well defined indole ring orientation and are therefore good candidates for use in in situ NMR. |
