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) approximately 2.1-15.2-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.
Tikunova
Svetlana B
SB
Department of Physiology and Cell Biology, The Ohio State University, Columbus, Ohio 43210, USA. tikunova1@hotmail.com
Davis
Jonathan P
JP
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000378:metabolism; D006801:Humans; D009154:Mutation; D009206:Myocardium / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics; D013329:Structure-Activity Relationship; D019209:Troponin C / Q000235:genetics* / Q000378:metabolism
D019209:Troponin C; D002118:Calcium