Characterization of a Cardiac Troponin C-Troponin I Chimera Protein

Association of the regulatory N-domain of cardiac troponin C (cNTnC) with the switch region of cardiac troponin I (cTnI147-163) is a major step in Ca2+ regulation of myocardial contraction by troponin as it triggers a series of downstream events that leads to contraction. Investigation of this interaction in the presence and absence of ligands, including Ca2+ and cardiotonic drugs, is important to understanding the regulation of cardiac contraction. This is commonly studied in vitro using isolated cNTnC and cTnI147-163; however, this does not accurately reflect the in situ environment since cNTnC and cTnI147-163 are spatially confined in the myofibril by being attached to their respective structural scaffold domains, the C-domain of cTnC (cCTnC) and the troponin T binding region of cTnI (cTnI80-136). We have designed, expressed and characterized a cardiac chimera (cChimera) protein containing cNTnC linked to cTnI144-173 through a linker containing a tobacco etch virus or thrombin protease cleavage site (similar to skeletal chimera of Tiroli et al, 2005). Our results show that Ca2+ binding to cChimera (KD 0.15 ± 0.05 μm) is tighter than to cNTnC (KD 2.6 ± 0.1 μm) and to that observed in skinned muscle fibers (KD 1.2 μm). We assessed binding of the calcium sensitizer dfbp-o, and desensitizer W7, and show that cChimera is particularly useful for studying and searching interactions of cNTnC-cTnI144-173 with potential agonists. Our cChimera also facilitates the production of the switch cTnI peptide using bacterial cultures by enzyme specific proteolysis after expression along with cNTnC. The dynamic properties of cChimera, along with Ca2+ and drug binding properties, make cChimera a convenient system for the investigation of regulation of troponin by small molecules that target the cTnC-cTnI interface.