Calcium-Induced Dimerization of Troponin C: Mode of Interaction and Use of Trifluoroethanol as a Denaturant of Quaternary Structure

Abstract

Protein aggregation can be a problem, especially as a large number of proteins become available for structural studies at fairly high concentrations using solution techniques such as NMR spectroscopy. The muscle regulatory protein troponin C (TnC) undergoes a calcium-induced dimerization at neutral pH with a dissociation constant for the dimerization of 0.4 mM at 20 degrees C. The present study indicates that the mode of dimerization involves the N-domain of one monomer interacting with the N-domain of another monomer. Addition of the solvent trifluoroethanol (TFE) to a concentration of 15%, v/v, results in a 10-fold increase in the dimer dissociation constant of calcium-saturated TnC (4 mM at 20 degrees C), making TnC predominantly a monomer for spectroscopic studies. Further, TFE, at the concentrations used herein, acts to perturb the quaternary structure of TnC without adversely affecting the secondary or tertiary structure as evidenced by minimal changes to its CD spectra and 1H, 13C, and 15N NMR chemical shifts.

Calcium-dependent Changes in the Flexibility of the Regulatory Domain of Troponin C in the Troponin Complex

“…The main difference between calculated and measured T 2 times is in the region between residues 104 and 125, comprising the loop in EF-hand site III and the following helix F. These residues have longer T 2 relaxation times than the rest of the molecule, which cannot be explained by anisotropy, and suggest that TnC site III is flexible in the complex. This was not observed for the C-domain of TnC complexed with TnI-(1-40) (45), and although in isolated TnC Lys-107 is very flexible (27), the remainder of site III is not. The increase in flexibility for site III in the complex is likely the result of the interactions between TnC and the other two subunits.…”

“…A uniformly deuterated deletion mutant of chicken skeletal muscle TnT (isoform TnT-3), corresponding to the T2 domain, was expressed and purified as described (25). 13 C, 15 N-and 2 H, 13 C, 15 N-labeled recombinant chicken skeletal TnC were expressed as described (27). After lysis by a French press, salts (CaCl 2 to 5 mM, MgCl 2 to 1 mM, NaCl 2 to 50 mM, and DTT to 1 mM) were added to the soluble fraction of the lysate, which was then applied to a phenyl-Sepharose column, previously equilibrated with 50 mM Tris⅐Cl, pH 7.5, 50 mM NaCl, 5 mM CaCl 2 , 1 mM MgCl 2 , 1 mM DTT, and 0.01% sodium azide.…”

“…A control sample (TnC⅐Ca 4 2ϩ ϩ TnI-(115-131)) was produced using the same buffer and conditions for the presence of calcium. To 2 H, , an equimolar amount of unlabeled TnI-(115-131) peptide was added to prevent the previously described dimerization of TnC through the N domain (27).…”

Solution secondary structure of calcium‐saturated troponin C monomer determined by multidimensional heteronuclear NMR spectroscopy

“…T2 data (Slupsky et al, 1995) suggest that the only flexible linker region is that between helices D and E, as well as F and G. The linker in the N-domain (between helices B and C) does not appear flexible on the time scale of the T2 experiment. Similar results were obtained for calmodulin, which has similar linker regions (Barbato et a]., 1992).…”

“…At the concentration required for NMR observation, the calcium-saturated state of TnC in water shows NMR resonance linewidths of the N-domain indicative of aggregation (Slupsky et al, 1995). Better linewidths can be achieved at high temperatures (Slupsky et al, 1995); however, many resonances were lost due to amide exchange with the solvent. Varying pH and salt concentrations had minimal effect on reducing the aggregation problem (C.M.…”