McTnI_29-Cys212 retained normal COOH-terminal conformation and binding affinities for TnC and TnT

The McTnI_29-Cys212 protein with substitutions of two internal Cys residues and addition of a Cys at the COOH-terminus (Fig. 1) was examined by epitope analysis using enzyme-linked immunosorbent assay (ELISA) for effect of the modifications on COOH-terminal conformation. The results in Fig. 3A demonstrated that McTnI_29-Cys212 gave an affinity titration curve for the anti-COOH-terminal segment mAb TnI-1 comparable to that of the McTnI_29–211 control, indicating retained wild type molecular conformation in the COOH-terminal end segment.

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Figure 3

Preserved conformation and function of McTnI_29-Cys212

A. Affinity titration using mAb TnI-1 recognizing a COOH-terminal epitope [11] showed a binding curve identical to the McTnI_29–211 control, indicating that the modifications in McTnI_29-Cys212 (Fig. 1A) did not alter the conformation of the COOH-terminal end segment. B. The TnC-binding curves showed that McTnI_29-Cys212 had a Ca²⁺-enhanced binding to TnC, similar to that of McTnI_29–211. C. The TnT-binding curves showed that McTnI_29-Cys212 bound TnT similarly to that of McTnI_29–211.

The functionality of McTnI_29-Cys212 was examined for its binding to TnC and TnT. Protein binding results in Fig. 3B showed that McTnI_29-Cys212 bound chicken fast TnC in a Ca²⁺-enhanced manner as anticipated [4] with same affinities as that of the McTnI_29–211 control. McTnI_29-Cys212 also bound bovine cardiac TnT same as that of McTnI_29–211 control (Fig. 3C). These data verified that the McTnI_29-Cys212 model molecule has retained normal biochemical activity, suitable for functional studies.

Calcium-regulated conformational change in the COOH-terminal end segment of TnI

Fluorescence image of SDS-gel (data not shown) verified that McTnI_29-Cys212 was effectively labeled with Alexa Fluor 532 dye at the COOH-terminal end Cys₂₁₂ through maleimide-thiol group reactions.

The fluorescently labeled McTnI_29-Cys212 was able to reconstitute tertiary troponin complex in vitro with chicken fast TnC and bovine cardiac TnT. Fluorescence image of SDS-gel (data not shown) further verified fluorescently labeled TnI in the troponin complex purified by gel filtration chromatography, which was used for fluorescence study of Ca²⁺-induced conformational changes in the COOH-terminal end segment of TnI.

As specified in the manufacturer’s datasheet, the emission spectral scans of Alexa Fluor 532 free dye detected a peak at 552 nm. The fluorescently labeled troponin complex showed a small red-shift of the emission peak to 558 nm (data not shown). The fluorescence emission peak did not change further during calcium titrations, indicating the 552 nm to 558 nm red-shift was due to the dye-protein conjugation.

Fluorescence intensity of the free dye exhibited mild decreases during the titration, reflecting a quenching effect. In contrast, TnI COOH terminus-labeled troponin complex showed increases in fluorescence intensity when [Ca²⁺] was increased during the titration (data not shown). The results indicated conformational changes in the COOH terminus of TnI when calcium binds to TnC in the troponin complex.

Ca²⁺ induced conformational responses in the COOH-terminal end segment of TnI

Plotted from the fluorescence spectral data, the TnI-COOH-terminus-labeled troponin complex demonstrated a sigmoid shape calcium titration curve (Fig. 4). EGTA back-titration from pCa 4 to pCa 8 showed that the effects of calcium were reversible.

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Figure 4

Calcium regulated conformational change in the COOH-terminal end segment of TnI

Calcium titration curve of TnI-COOH-terminus-labeled troponin complex was plotted from the fluorescent intensity data after correction for the dye quenching effect. Calcium titration of secondary structure of free TnC was performed using far-UV CD spectral analysis. The molar ellipticity trough values at 222 nm indicating increases in α-helical contents in TnC when calcium is bound were collected to plot the calcium titration curve. Both curves were corrected for dilution factors, normalized to the maximal change, and fitted to the Hill equation (see Experimental Procedures). The open symbols represent EGTA back-titration from pCa 4 to 8, demonstrating the reversibility of the calcium-induced conformational changes in TnI COOH-terminal end segment and TnC. The results demonstrated very similar shapes of the calcium responding curves for the molecular conformations of TnI COOH-terminal end segment and TnC with pCa₅₀ values of 6.10±1.02 and 6.32±1.19, respectively, with no statistical difference.

As a control, Ca²⁺-induced conformational changes in free TnC was titrated using far-UV CD spectral analysis and compared with the fluorescence titration of TnI-COOH-terminus-labeled troponin complex using the same set of pCa buffers. Consistent with published data [28,29], the far-UV CD spectra of TnC showed that Ca²⁺-binding increased the α-helix content of TnC as shown by the decreases in molar ellipticity. The sigmoid shape calcium titration curve of TnC derived from far-UV CD measurements (Fig. 4) was similar to that previously reported [28,29].

Superimposing the calcium titration curves for the fluorescence intensity of TnI-COOH-terminus-labeled troponin and the CD spectra of free TnC demonstrated similar patterns of calcium responsiveness (Fig. 4).

The binding with TnI was previously shown to increase the Ca²⁺ affinity of TnC at the high affinity sites III and IV (30). However, our assay buffer contained 3 mM Mg²⁺ to minimize this effect. Therefore, although the Ca²⁺ titration curves of free TnC and TnI COOH-terminal fluorescence represented individual conformational changes, their similar shapes suggested that the COOH-terminal end segment of TnI underwent conformational changes corresponding to the binding of Ca²⁺ to TnC in a troponin complex, supporting a physiological significance.

COOH-terminal truncated TnI

The cDNA template encoding McTnI_29–211 was modified by PCR to produce a COOH-terminal truncated TnI (McTnI_29–192) [38]. The McTnI_29–192 protein was expressed in E. coli, purified as above, and verified by Western blotting using mAb 4H6 that recognizes a middle region epitope [16].

Engineering a mini-TnT lacking the tropomyosin-binding sites

A cDNA corresponding to the exons 15–17-encoded COOH-terminal segment of mouse cardiac TnT (McTnT_248–305) was constructed using PCR from the full length cDNA and inserted into pAED4 plasmid for non-fusion protein expression. As shown in the crystal structures of troponin [9,10], this fragment of TnT fully retains the binding sites for TnI and TnC (Fig. 1B) and, therefore, can be used to reconstitute tertiary troponin complex.

After DNA sequencing to verify the expression plasmid, McTnT_248–305 protein was expressed in E. coli culture. BL21(DE3)pLysS E. coli cells transformed with the recombinant plasmid were cultured, induced in the early log phase, and harvested. The cells were lysed in 5 mM EDTA, 15 mM β-mercaptoethanol, 0.5 mM phenylmethylsulfonyl fluoride, 50 mM Tris-HCl, pH 8.0, using a French press. The protein extract was fractionated with ammonium sulfate precipitation at 0°C. The fraction between 30–50% saturation was dialyzed against 0.1 mM EDTA, brought to 6 M urea, 15 mM β-mercaptoethanol, 0.5 mM phenylmethylsulfonyl fluoride, 20 mM imidazole, pH 7.0, clarified by centrifugation, and loaded on a CM52 column equilibrated with the same buffer. The column was eluted with 0–500 mM linear KCl gradient, and the protein peaks were analyzed with SDS-PAGE. The fractions containing McTnT_248–305 were dialyzed against 0.1 mM EDTA and concentrated by lyophilization for further purification using Sephadex G75 gel filtration chromatography in 6 M urea, 0.5 M KCl, 0.1 mM EDTA, 6 mM β-mercaptoethanol, 0.1 mM phenylmethylsulfonyl fluoride, 10 mM imidazole, pH 7.0. Protein peaks were analyzed by SDS-PAGE and the fractions containing pure McTnT_248–305 were dialyzed against de-ionized water and lyophilized.

Preparation of other proteins

Bovine cardiac TnT was purified from adult left ventricular muscle as previously described [39]. Chicken fast TnC was purified from E. coli expression as described previously [40]. Bovine cardiac α-tropomyosin was purified from adult ventricular muscle as described [41].

mAb epitope analysis

Based on the nature that antibody-antigen bindings are dependent on fits between 3D protein structures [42], mAb TnI-1 that recognizes a conserved epitope in the COOH-terminal end segment of TnI [11] was used as a 3D structural probe to examine the modified TnI molecules.

ELISA epitope analysis [42,43] was applied to detect whether the C81I and C98S substitutions and the addition of a Cys to the COOH terminus had any effect on the molecular conformation of the COOH-terminal end segment of TnI. McTnI_29–211 and McTnI_29-Cys212 were dissolved at 1 μg/mL in Buffer A (20 mM PIPES, 3 mM MgCl₂, 100 mM KCl, pH 7.0) and coated separately in 96-well microtitering plates (100 μL/well) at 4°C overnight. After washing with Buffer T (Buffer A plus 0.05% Tween-20) to remove unbound protein, the immobilized TnI was incubated with serial dilutions of mAb TnI-1 in Buffer B (Buffer T plus 0.1% bovine serum albumin (BSA)) at room temperature for 2 hr. The plates were then processed with a standard ELISA procedure including Buffer T washes, horseradish peroxidase-conjugated anti-mouse immunoglobulin second antibody (Santa Cruz Biotechnology) incubation, and H₂O₂/2,2′-azinobis(3-ethylbenzothiazolinesulfonic acid) substrate reaction. Absorbance at 415 nm of each assay well was recorded at a series of time points during the substrate development using an automated microplate reader (Bio-Rad Benchmark). The A_415nm values in the linear course of the color development were plotted to obtain mAb affinity curves that reflect the molecular conformation at the TnI-1 epitope in the COOH-terminal end segment of TnI. The experiments were performed in triplicate and repeated.

Protein binding assays

ELISA solid-phase protein binding experiments were performed to examine the interactions of McTnI_29-Cys212 with TnT and TnC. Purified McTnI_29–211 and McTnI_29-Cys212 proteins were dissolved at 5 μg/mL in Buffer A containing 1 mM dithiothreitol (DTT) and non-covalently immobilized onto 96-well microtitering plates. After removing free protein and blocking the wells with 1% BSA in Buffer T, the immobilized TnI was incubated with serial dilutions of bovine cardiac TnT or chicken fast TnC in Buffer B containing 1 mM DTT. After Buffer T washes once with DTT and four times without DTT, the TnT or TnC bound to the immobilized TnI was quantified via an anti-TnT mAb CT3 [38] or an anti-TnC mAb 2C3 [40], respectively. The plates were then processed with ELISA procedure as described above to obtain binding affinity curves for TnT and TnC. The experiments were performed in triplicate and repeated.

Fluorescence labeling of McTnI_29-Cys212

Purified McTnI_29-Cys212 protein was dissolved in 100 mM KCl, 10 mM imidazole-HCl, pH 7.0, and mixed with Alexa Fluor 532 C5-maleimide dye in a protein:dye molar ratio of 1:10 for incubation with shaking at room temperature for 2 hr. The labeling mix was put through a Superose F12 column (GE Healthcare) to remove free dye. McTnI_29-Cys212 labeled at the COOH-terminal Cys₂₁₂ was examined by SDS-PAGE and fluorescence imaging using a Typhoon 9210 scanner (GE Healthcare) with excitation at 532 nm and recording the emission at 555 nm. Total protein in the gels was revealed with Coomassie Brilliant Blue R-250 staining.

Reconstitution of troponin complex

Tertiary troponin complex was reconstituted in vitro using a) McTnI_29–211, McTnI_29–192 or fluorescence-labeled McTnI_29-Cys212, b) intact bovine cardiac TnT or McTnT_248–305, and c) chicken fast TnC. The protein mixture was dissolved in 6 M urea, 1 M KCl, 1 mM DTT, 10 mM imidazole-HCl, pH 7.1, and dialyzed to gradually remove urea and high salt to reach the final assay condition of 100 mM KCl, 3 mM MgCl₂, 10 mM imidazole-HCl, pH 7.1. To compensate for the predicted higher loss of McTnT_248–305 and TnC during dialysis due to their smaller sizes, these two components were used in 2-fold and 1.4-fold molar excesses, respectively. The reconstituted troponin complex was purified using Superose F12 column in the assay buffer. The eluted troponin peak containing the three troponin subunits was identified by SDS-PAGE for use in structure-function studies.

Fluorescence measurements

Steady-state fluorescence measurements were carried out at room temperature using an ISS PC1 spectrofluorometer with the band pass of the excitation and emission monochromators both at 8 nm. Fluorescence intensity was recorded with the excitation at 528 nm for the TnI-COOH-terminus labeled troponin complex and Alexa Fluor 532 free dye control.

Far-UV CD measurements

Chicken fast TnC was dissolved at 0.5 mg/mL in 100 mM KCl, 3 mM MgCl₂, and 10 mM sodium phosphate, pH 7.1. Far UV CD spectra of TnC were measured on a Jasco J715 spectropolarimeter at room temperature following a previously described method [28].

Calcium titration and fluorescence curve fitting

To avoid metal ion contaminations, all solutions were prepared with Milli-Q deionized water in HCl pre-treated plastic ware. Stocks of 10 and 20 mM CaCl₂ were prepared from 1 M CaCl₂ standard purchased from Fluka. Titrations were conducted by adding the CaCl₂ stocks in serial increments. With 10 mM imidazole buffer in the fluorescence measurements of troponin complex and 10 mM sodium phosphate buffer in the far-UV CD measurements of TnC, no change in pH was detected before and after the calcium titrations. Free Ca²⁺ concentrations buffered by 1 mM EGTA were calculated using Maxchelator program (http://maxchelator.stanford.edu). pCa values were calculated as -log(free [Ca²⁺]).

For the TnI-COOH-terminus-labeled troponin complex, the fluorescence intensity at 558 nm was recorded and corrected by the dilution factor during the titration and the quenching rate of the free dye (Equation 1).

In far-UV CD calcium titration of TnC, the molar ellipticity at 222 nm was recorded and corrected by the dilution factor (Equation 2).

The corrected fluorescence intensity or molar ellipticity was normalized to the percentage of their maximal changes using Equation 3 and plotted against the pCa values.

Curve fitting for the titration data was performed with nonlinear Hill equation (Equation 4) using the Origin software.

in which Y represents the normalized fluorescence intensity or molar ellipticity, K is the apparent dissociation constant, and n is the Hill coefficient.

Detection of Ca²⁺-regulated binding between the COOH-terminal end segment and tropomyosin

Similar to the binary protein binding experiments described above, ELISA experiments were carried out to investigate the binding between TnI COOH-terminal end segment and tropomyosin. Reconstituted tertiary troponin complex containing McTnT_248–305 and McTnI_29–211 or McTnI_29–192 were immobilized onto microtitering plates in Buffer A containing DTT at pCa 4 [40]. After washing and blocking the wells as above but at pCa 4, the immobilized troponin were incubated with serial dilutions of tropomyosin in Buffer B at pCa 4 or pCa 9. The plates were then processed as above with washing, incubation with anti-tropomyosin mAb CH1 (a gift from Dr. Jim Lin, University of Iowa), washing to remove excess CH1, incubation with horseradish peroxidase-second antibody, and final washes, in which the pCa remained to be controlled at 4 or 9 to examine the effect of Ca²⁺. After substrate color development, tropomyosin-binding curves were plotted with data from triplicate wells and repeated experiments. Immobilized intact mouse cardiac TnT alone and McTnT_248–305 alone were examined as controls in the tropomyosin-binding experiments.

This study was supported by grants from the National Institutes of Health AR-048816 and HL-098945.

We thank Stephen Chong for participating in the construction of cTnI_29–192, Annie Wang for technical assistance, Dr. Jim Lin for the CH1 mAb, and the Keck Biophysics Facility at Northwestern University for instrumental services.