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.
Highlights
Vertebrate striated muscle contraction is regulated by Ca2ϩ, and the proteins that mediate this regulation in the contractile muscle are tropomyosin (Tm)1 and troponin (Tn)
Residues [193–238] of Fast skeletal TnT (fsTnT) hydrophobic repeats (HRs) and residues 58 –103 of fsTnI HR correspond to the amino acid residues 226 –271 and 90 –135 of the cardiac troponin T (TnT) and troponin I (TnI) isoform, respectively, which are observed to form a heterodimeric coiled-coil in the crystal structure of the human cardiac troponin core complex (21)
We have previously shown that mutations in the HR domain of fsTnT lead to a lack of or a strong decrease in interaction with TnI HR (18), suggesting that the HR domains are involved in a coiled-coil formation
Summary
Materials—Common reagents and buffer components were purchased from Sigma unless mentioned otherwise. 15 ml of the peptide solution was packed into a Spectrapore dialysis bag and dialyzed for 10 h stepwise in three buffers containing 20 mM Tris-HCl, pH 7.5, 300 mM NaCl, and [4, 2], and 0 M urea, respectively. The Tm values were checked by differentiating the data with respect to temperature and fitting it to the equation after application of a three-point smoothing window, Stabilization of the Fast Skeletal TnT-TnI Coiled-coil. Samples were dialyzed overnight at 4 °C in a 10 mM Tris-HCl, 300 NaCl, 1 mM ME, pH 7.5 buffer before the experiments. Data analysis was performed using the least square method with the Origin-based software provided by the manufacturer to determine the equilibrium binding constant (KA), the enthalpy of the complex formation (⌬H), and the entropy (⌬S) of the reaction.
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