Designing calcium-sensitizing mutations in the regulatory domain of cardiac troponin C

Cardiac troponin C belongs to the EF-hand superfamily of calcium-binding proteins and plays an essential role in the regulation of muscle contraction and relaxation. To follow calcium binding and exchange with the regulatory N-terminal domain (N-domain) of human cardiac troponin C, we substituted Phe at position 27 with Trp, making a fluorescent cardiac troponin C-F27W. Trp(27) accurately reported the kinetics of calcium association and dissociation of the N-domain of cardiac troponin C-F27W. To sensitize the N-domain of cardiac troponin C-F27W to calcium, we individually substituted the hydrophobic residues Phe(20), Val(44), Met(45), Leu(48), and Met(81) with polar Gln. These mutations were designed to increase the calcium affinity of the N-domain of cardiac troponin C by facilitating the movement of helices B and C (BC unit) away from helices N, A, and D (NAD unit). As anticipated, these selected hydrophobic residue substitutions increased the calcium affinity of the regulatory domain of cardiac troponin C-F27W similar to2.1-15.2-fold. Surprisingly, the increased calcium affinity caused by the hydrophobic residue substitutions was largely due to faster calcium association rates (2.6-8.7-fold faster) rather than to slower calcium dissociation rates (1.2-2.9-fold slower). The regulatory N-domains of cardiac troponin C-F27W and its mutants were also able to bind magnesium competitively and with physiologically relevant affinities (1.2-2.7 mM). The design of calcium-sensitizing cardiac troponin C mutants presented in this work enhances the understanding of how to control cation binding properties of EF-hand proteins and ultimately their structure and physiological function.