Microtiter plate monoclonal antibody epitope analysis of Ca2+- and Mg2+-induced conformational changes in troponin C

Abstract

Spectroscopic methods such as circular dichroism and Förster resonance energy transfer are current approaches for monitoring protein conformational changes. Those analyses require special equipment and expertise. The need for fluorescence labeling of the protein may interfere with the native structure. We have developed a microtiter plate-based monoclonal antibody (mAb) epitope analysis to detect protein conformational changes in a high throughput manner. This method is based on the concept that the affinity of the antigen-binding site of an antibody for the specific antigenic epitope will change when the 3-D structure of the epitope changes. The effectiveness of this approach was demonstrated in the present study on troponin C (TnC), an allosteric protein in the Ca(2+) regulatory system of striated muscle. Using TnC purified by a highly effective rapid procedure and mAbs developed against epitopes in the N- and C-domains of TnC enzyme-linked immunosorbant assay (ELISA) clearly detected Ca(2+)-induced conformational changes in both the N-terminal regulatory domain and the C-terminal structural domain of TnC. On the other hand, Mg(2+)-binding to the C-domain of TnC resulted in a long-range effect on the N-domain conformation, indicating a functional significance of Ca(2+)-Mg(2+) exchange at the C-domain metal ion-binding sites. In addition to further understanding of the structure-function relationship of TnC, the data demonstrate that the mAb epitope analysis provides a simple high throughput method for monitoring 3-D structural changes in native proteins under physiological condition and has broad applications in protein structure-function relationship studies.

Fig. 1. ELISA epitope analysis

This flowchart illustrates the ELISA-based epitope analysis using a mAb. The protein to be analyzed (TnC in this example) is non-covalently coated at random orientations onto the plastic surface in a 96-well microtiter plate. The coating buffer can apply various ionic, pH, and other conditions as a pre-treatment of the protein to be studied. After washing to remove unbound TnC, remaining free surface area in the assay wells is blocked by treatment with non-ionic detergent (Tween-20). A specific anti-TnC antibody will be added to the plate and incubated with the immobilized target protein (TnC) under buffer conditions that are the same as or different from that of the coating buffer. After incubation, the unbound antibody will be removed by washes under desired stringency and buffer conditions. The plate will then be incubated with enzyme-conjugated second antibody, washed again to remove unbound second antibody, and incubated with a colorimetric substrate of the enzyme. The binding affinity between the target protein and the first antibody will be quantified by enzymatic colorimetric reaction and recorded using a microplate reader.

Fig. 2. Expression of recombinant TnC in E. coli and rapid purification

The SDS-PAGE gels show the bacterial expression and purification profile of mouse cardiac TnC. The TnC protein recovered in the total bacterial lysate was effectively enriched by ammonium sulfate fractionation between 80-100% saturation and purified by sizing fractionation on a Sephadex G-75 sizing column. The same procedure has been reproduced for the purifications of human cardiac TnC and chicken fast TnC (data not shown).

Fig. 3. Anti-TnC mAbs

(A) The illustration shows a linear structure map of TnC with the N-domain and C-domain fragments aligned. AA, number of amino acids; MW, molecular weight; pI, isoelectric point. (B) Total protein extracts from chicken breast and mouse extensor digitorum longus (EDL) muscles were resolved by SDS-PAGE and examined by Western blotting using mAbs 4E7, 2C3, 2D10 and 2A7. The results show that all four mAbs recognized the immunogen, chicken fast TnC and only 2C3 reacted weakly to mouse fast TnC. The higher molecular weight bands weakly recognized by mAbs 4E7, 2C3 and 2D10 are likely myosin light chains that are proteins homologous to TnC [6]. (C) Intact mouse cardiac TnC, intact chicken fast TnC, chicken fast TnC N-domain and chicken fast TnC C-domain were analyzed by Tris-Tricine SDS-PAGE and Western blotting. The results showed that these mAbs raised against fast TnC do not cross react with cardiac TnC. The epitopes recognized by mAbs 4E7 and 2C3 are in the N-domain fragment and that of 2D10 and 2A7 are in the C-domain fragment. The higher molecular weight band detected in the TnC C-domain fragment sample (indicated by the *) was likely aggregated dimmers, although a sufficient amount of reducing agent was present in the SDS-gel sample buffer.

Fig. 4. Ca²⁺- and Mg²⁺-induced conformational change in the N-domain of TnC detected by ELISA epitope analysis

(A) The ELISA titration curves showed that the affinity of the anti-N-domain mAb 4E7 for intact TnC was significantly higher in the presence of Ca²⁺ (pCa 4) than that at pCa 9 (**P<0.01), indicating a Ca²⁺-induced conformational change involving the mAb 4E7 epitope in the N-domain of TnC. The presence of 3 mM free Mg²⁺ at pCa 9 also resulted in an increase of the binding affinity of mAb 4E7 (**P<0.01), though in a less degree than the Ca²⁺ effect. However, 3 mM free Mg²⁺ at pCa 4 did not produce any additive effect on the binding affinity of mAb 4E7. (B) When the isolated N-domain fragment of TnC was tested for mAb 4E7 affinity in the presence or absence of Ca²⁺ and/or Mg²⁺, the titration curves demonstrated that Ca²⁺ binding-induced a conformational change in the isolated N-domain (**P<0.01) similar to that in the intact TnC. 3 mM free Mg²⁺, however, produce a detectable (*P<0.05) but much smaller change in the isolated N-domain than that in intact TnC. The results suggest that Mg²⁺ affects the N-domain conformation of intact TnC mainly through binding to the C-domain sites.

Fig. 5. Ca²⁺-induced conformational change in the C-domain of TnC detected by ELISA epitope analysis

(A) The ELISA titration curves showed that the affinity of the anti-C-domain mAb 2D10 for intact TnC was significantly lower in the presence of Ca²⁺ (pCa 4) than that at pCa 9 (**P<0.01), indicating a Ca²⁺-induced conformational change involving the mAb 2D10 epitope in the C-domain of TnC. The presence of 3 mM free Mg²⁺ alone (at pCa 9) did not result in significant change in the binding affinity of mAb 2D10 or any additive effect in the presence of Ca²⁺ (at pCa 4). (B) When the isolated C-domain fragment of TnC was tested for affinity to mAb 2D10, the results showed an almost identical Ca²⁺ effect to that on intact TnC (**P<0.01) and Mg²⁺ remained no significant effect. The results suggest that Ca²⁺ binding to the C-domain sites of TnC produces a conformational change that is absent upon the binding of Mg²⁺.