Fluorescent Protein-Based Ca(2+) Sensor Reveals Global, Divalent Cation-Dependent Conformational Changes in Cardiac Troponin C

Cardiac troponin C (cTnC) is a key effector in cardiac muscle excitation-contraction coupling as the Ca(2+) sensing subunit responsible for controlling contraction. In this study, we generated several FRET sensors for divalent cations based on cTnC flanked by a donor fluorescent protein (CFP) and an acceptor fluorescent protein (YFP). The sensors report Ca(2+) and Mg(2+) binding, and relay global structural information about the structural relationship between cTnC’s N- and C-domains. The sensors were first characterized using end point titrations to decipher the response to Ca(2+) binding in the presence or absence of Mg(2+). The sensor that exhibited the largest responses in end point titrations, CTV-TnC, (Cerulean, TnC, and Venus) was characterized more extensively. Most of the divalent cation-dependent FRET signal originates from the high affinity C-terminal EF hands. CTV-TnC reconstitutes into skinned fiber preparations indicating proper assembly of troponin complex, with only ~0.2 pCa unit rightward shift of Ca(2+)-sensitive force development compared to WT-cTnC. Affinity of CTV-TnC for divalent cations is in agreement with known values for WT-cTnC. Analytical ultracentrifugation indicates that CTV-TnC undergoes compaction as divalent cations bind. C-terminal sites induce ion-specific (Ca(2+) versus Mg(2+)) conformational changes in cTnC. Our data also provide support for the presence of additional, non-EF-hand sites on cTnC for Mg(2+) binding. In conclusion, we successfully generated a novel FRET-Ca(2+) sensor based on full length cTnC with a variety of cellular applications. Our sensor reveals global structural information about cTnC upon divalent cation binding.