title: Identification of a region of fast skeletal troponin T required for stabilization of the coiled-coil formation with troponin I.
abstract: We have previously identified evolutionarily conserved heptad hydrophobic repeat (HR) domains in all isoprotein members of troponin T (TnT) and troponin I (TnI), two subunits of the Ca(2+)-regulatory troponin complex. Our suggestion that the HR domains are involved in the formation of a coiled-coil heterodimer of TnT and TnI has been recently confirmed by the crystal structure of the core domain of the human cardiac troponin complex. Here we studied a series of recombinant deletion mutants of the fast skeletal TnT to determine the minimal sequence required for stable coiled-coil formation with the HR domain of the fast skeletal TnI. Using circular dichroism spectroscopy, we measured the alpha helical content of the coiled-coil formed by the various TnT peptides with TnI HR domain. Sedimentation equilibrium experiments confirmed that the individual peptides of TnT were monomeric but formed heterodimers when mixed with HR domain of TnI. Isothermal titration calorimetry was then used to directly measure the affinity of the TnT peptides for the TnI HR domain. Surprisingly we found that the HR regions alone of the fast skeletal TnT and TnI, as defined earlier, were insufficient to form a coiled-coil. Furthermore we showed that an additional 14 amino acid residues N-terminal to the conserved HR region (TnT residues 165-178) are essential for the stable coiled-coil formation. We discuss the implication of our finding in the fast skeletal troponin isoform in the light of the crystal structure of the cardiac isoform.
authors.affiliation: Program in Cell, Molecular and Developmental Biology, Sackler School of Graduate Biomedical Sciences, Tufts University School of Medicine, Boston, MA 02111, USA.
authors.forename: Subhradip
authors.initials: S
authors.lastname: Mukhopadhyay
authors.forename: Knut
authors.initials: K
authors.lastname: Langsetmo
authors.forename: Walter F
authors.initials: WF
authors.lastname: Stafford
authors.forename: Gillian D
authors.initials: GD
authors.lastname: Henry
authors.forename: James D
authors.initials: JD
authors.lastname: Baleja
authors.forename: Satyapriya
authors.initials: S
authors.lastname: Sarkar
mesh_terms: D006801:Humans; D008958:Models, Molecular; D018656:Muscle Fibers, Fast-Twitch / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D019210:Troponin I / Q000737:chemistry* / Q000378:metabolism; D020107:Troponin T / Q000737:chemistry* / Q000378:metabolism
chemical_list: D010446:Peptide Fragments; D019210:Troponin I; D020107:Troponin T