The E41A mutation in skeletal troponin C reduced Ca2+ binding affinity of site I by approximately 100-fold and of site II by approximately 10-fold compared to wild-type.
NMR studies reveal that modifications in Ca2+-binding site I of troponin C critically affect the energetics and mechanism of Ca2+-induced structural changes.
Ca2+ binding to the N-domain of skeletal muscle troponin C (sNTnC) induces an "opening" of the structure Gagné, S. M. , et al. (1995) Nat. Struct. Biol. 2, 784-789, which is typical of Ca2+-regulatory proteins. However, the recent structures of the E41A mutant of skeletal troponin C (E41A sNTnC) Gagné, S. M. , et al. (1997) Biochemistry 36, 4386-4392 and of cardiac muscle troponin C (cNTnC) Sia, S. K. , et al. (1997) J. Biol. Chem. 272, 18216-18221 reveal that both of these proteins remain essentially in the "closed" conformation in their Ca2+-saturated states. Both of these proteins are modified in Ca2+-binding site I, albeit differently, suggesting a critical role for this region in the coupling of Ca2+ binding to the induced structural change. To understand the mechanism and the energetics involved in the Ca2+-induced structural transition, Ca2+ binding to E41A sNTnC and to cNTnC have been investigated by using one-dimensional 1H and two-dimensional 1H, 15N-HSQC NMR spectroscopy. Monitoring the chemical shift changes during Ca2+ titration of E41A sNTnC permits us to assign the order of stepwise binding as site II followed by site I and reveals that the mutation reduced the Ca2+ binding affinity of the site I by approximately 100-fold from KD2 = 16 microM [sNTnC; Li, M. X. , et al. (1995) Biochemistry 34, 8330-8340 to 1. 3 mM (E41A sNTnC) ] and of the site II by approximately 10-fold from KD1 = 1. 7 microM (sNTnC) to 15 microM (E41A sNTnC). Ca2+ titration of cNTnC confirms that cNTnC binds only one Ca2+ with a determined dissociation constant KD of 2. 6 microM. The Ca2+-induced chemical shift changes occur over the entire sequence in cNTnC, suggesting that the defunct site I is perturbed when site II binds Ca2+. These measurements allow us to dissect the mechanism and energetics of the Ca2+-induced structural changes.
Li et al. (Wed,) reported a other. E41A mutation in skeletal troponin C vs. Wild-type skeletal muscle troponin C (sNTnC) was evaluated on Ca2+ binding affinity (dissociation constant KD). The E41A mutation in skeletal troponin C reduced Ca2+ binding affinity of site I by approximately 100-fold and of site II by approximately 10-fold compared to wild-type.