Troponin-C mutants with increased calcium affinity.
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.
da Silva
A C
AC
Departamento de Bioquímica, Universidade de Sao Paulo, Brazil.
de Araujo
A H
AH
Herzberg
O
O
Moult
J
J
Sorenson
M
M
Reinach
F C
FC
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D066298:In Vitro Techniques; D009119:Muscle Contraction / Q000502:physiology; D009132:Muscles / Q000378:metabolism / Q000502:physiology; D009154:Mutation; D011487:Protein Conformation; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014336:Troponin / Q000235:genetics / Q000378:metabolism*; D019209:Troponin C
D011994:Recombinant Proteins; D014336:Troponin; D019209:Troponin C; D002118:Calcium