Microtiter plate monoclonal antibody epitope analysis of Ca2+- and Mg2+-induced conformational changes in troponin C.
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.
Jin
Jian-Ping
JP
Section of Molecular Cardiology, Evanston Northwestern Healthcare and Northwestern University, Feinberg School of Medicine, 2650 Ridge Avenue, Evanston, IL 60201, USA. jpjin@northwestern.edu
Chong
Stephen M
SM
Hossain
M Moazzem
MM
D000911:Antibodies, Monoclonal / Q000737:chemistry*; D002118:Calcium / Q000737:chemistry*; D018604:Epitope Mapping / Q000379:methods*; D008274:Magnesium / Q000737:chemistry*; D008832:Microchemistry / Q000379:methods; D011487:Protein Conformation; D017186:Titrimetry / Q000379:methods; D019209:Troponin C / Q000737:chemistry* / Q000648:ultrastructure*
D000911:Antibodies, Monoclonal; D019209:Troponin C; D008274:Magnesium; D002118:Calcium