Troponin‐C mutants with increased calcium affinity

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Abstract
Binding of two Ca2+ to the regulatory sites I and II of troponin C (TnC) induces a conformational transition believed to be responsible for the activation of muscle contraction. Based on the known crystal structure (2Ca2+ state), a model for the transition to the 4Ca2+ state has been proposed [Herzberg, O., Moult, J. & James, M. N. G. (1986) J. Biol. Chem. 261, 2638–2644]. The proposed conformational transition predicts that during Ca2+ binding a number of nonpolar residues become exposed to the solvent, creating a hydrophobic patch. Such a model implies that mutation of the hydrophobic to polar residues should increase the Ca2+ affinity at the regulatory sites and reduce the Ca2+ concentration necessary for muscle activation. To test this prediction, we have constructed and functionally characterized two troponin‐C mutants (V45T and M48A mutations).Direct calcium‐binding measurements in the mutants demonstrate an increase in the Ca2+ affinity for two low‐affinity sites. Replacement of endogenous troponin C in skinned muscle fibers by TnC with mutations V45T or M48A increased the Ca2+ sensitivity of their tension development. These results show that the model can be used to construct mutants that regulate muscle contraction at lower Ca2+ concentrations. They provide further experimental support for the proposed calcium‐induced conformational change of troponin C and suggest that the predicted transition plays a central role in the activation of the thin filament.