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

The changes in protein three-dimensional (3-D)1 structure are commonly examined by spectroscopic methods such as circular dichroism and Forster resonance energy transfer (FRET). These methods need to be performed in expert laboratories and require highly specialized equipment. Spectroscopic methods often require external labeling of the protein, which may interfere with the native structure. Therefore, a more readily accessible method would be desirable for detecting changes in protein 3-D structure. The binding affinity between the paratope (the antigen binding site formed by the variable regions of immunoglobulin heavy and light chains) of an antibody and the specific epitope on a protein recognized by the antibody is dependent on the fit between the 3-D structures of the two proteins. Based on this concept, antibodies provide not only tools to identify specific proteins but also probes to detect and quantify changes in protein 3-D structure. The hybridoma technology has allowed the production of homogeneous monoclonal antibodies (mAb) [1] that recognize specific epitope structures on almost any given protein, generating site-specific probes for protein 3-D structural analysis. The infinite production of a mAb from the hybridoma cell line also allows the establishment of standardized probes for protein structural studies. Enzyme-linked immunosorbant assay (ELISA) is a rapid method to quantitatively determine antibody-antigen interactions [2]. Microtiter plate-based ELISA, like other solid phase immunological assays, applies repeated washes as the separation procedure and, therefore, is highly sensitive in detecting changes in antigen-antibody binding affinity to provide a quantitative measurement of the 3-D structural fit between the antigenic epitope and the antibody paratope. Together with its high throughput nature, microtiter plate-based ELISA can be readily employed for the epitope analysis of protein 3-D structures. Troponin C (TnC) is the Ca2+-binding subunit of troponin and an allosteric protein that plays a key function in regulating muscle contraction [3,4]. During the activation of striated muscles, cytoplasmic Ca2+ rises, binds to TnC, and induces a series of conformational changes in the troponin complex and the actin thin filament to activate the actomyosin ATPase and the development of force [5]. Two homologous TnC genes (fast skeletal muscle TnC and cardiac/slow skeletal muscle TnC) have evolved in vertebrates to encode the muscle-type-specific TnC isoforms [6]. Primary structures of the two TnC isoforms are largely conserved. X-ray crystallography and NMR spectroscopy have revealed the 3-D structure of TnC under various experimental conditions [7-9]. The structure of TnC is defined as the N-terminal regulatory domain and the C-terminal structural domain. A total of four E-F hand type divalent metal ion-binding sites are present in TnC. The C-domain of TnC contains two high affinity metal binding sites (sites III and IV) that bind Ca2+ (Kd of ∼107 M) and Mg2+ (Kd of ∼103 M) competitively. The N-domain of TnC contains two low affinity metal binding sites (Sites I and II) that bind only Ca2+ with Kd values of ∼16 μM and ∼1.7 μM, respectively, under the physiological Mg2+ concentrations [10,11]. The main structural difference between fast TnC and cardiac/slow TnC is that the N-domain Site I is inactive in cardiac/slow TnC [4]. The dynamic Ca2+-binding to the N-domain of TnC is the initial regulatory step of muscle contraction [12]. Based on the metal ion regulation of the allosteric protein TnC, we evaluated the application of ELISA epitope analysis using anti-TnC mAbs for detecting changes in the 3-D structure of TnC upon the binding of Ca2+ and/or Mg2+. The results showed a sensitive detection of Ca2+-induced conformational changes in both the N-terminal regulatory domain and the C-terminal structural domain of TnC. Mg2+-binding to the C-domain of TnC resulted in a long-range effect on the N-domain conformation, indicating a functional significance of Ca2+-Mg2+ 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.