Calmodulin (CaM) plays an important role in calcium (Ca2+)-dependent signal transduction. Ca2+ binding to CaM triggers a conformational change, forming a hydrophobic patch that is important for targeting protein recognition. Previously, we have characterized chimera proteins constructed by exchanging EF-hands of CaM with those of Troponin C (TnC) to be used as an informative probe to better understand the functionality of each EF-hand. Our results revealed that Ca2+ binding to chimeras impairs their hydrophobicity as determined from ANS binding. Such conformational changes altered their interaction with a CaM-binding domain (CMBD) peptide of Orai. Here we aim to measure additional thermodynamic parameters to better understand the conformational change upon Ca2+ binding using isothermal titration calorimetry (ITC). We determined the proton release upon Ca2+ binding. Our data indicates that approximately 0.3-0.7 protons are released to the buffer when CaM and chimeras are titrated with saturating Ca2+, suggesting that key residue(s) involved in Ca2+ binding are not altered by the exchanged EF-hands. The heat capacity change of Ca2+ binding (DCp) for CaM was determined to be ~ -0.66 kcal K-1 mol-1, generally consistent with reported values. The DCp values of 1TnC and 2TnC (1st and 2nd EF-hand exchange position respectively) are similar to CaM. On the other hand, 3TnC and 4TnC have very small DCp values ranging +0.004 to -0.17 kcal K-1 mol-1. The results suggest cooperative binding exists in domains as an exchanged EF affects the other EF within the same domain. Circular Dichroism revealed the secondary structures of apo- 3TnC and 4TnC resemble apo-CaM while those of holo- 1TnC and 2TnC resemble holo-CaM, indicating Ca2+ induces less helical change for 3TnC and 4TnC. The solvent accessible area changes determined from homology modeling and unfolding heat enthalpy change for each construct will be discussed to understand their structural changes upon Ca2+ binding.

Ca2+ induced conformational changes of calmodulin and troponin C chimera

253rd National Meeting (2017)

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