Open AccessTroponin‐C mutants with increased calcium affinity
Author(s) -
SILVA Ana Claudia R.,
ARAUJO Alexandre H. B.,
HERZBERG Osnat,
MOULT John,
SORENSON Martha,
REINACH Fernando C.
Publication year - 1993
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1993.tb17799.x
Subject(s) - troponin c , mutant , chemistry , calcium , biophysics , troponin , transition (genetics) , conformational change , binding site , biochemistry , stereochemistry , biology , medicine , organic chemistry , myocardial infarction , gene
Binding of two Ca 2+ 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 (2Ca 2+ state), a model for the transition to the 4Ca 2+ 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 Ca 2+ 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 Ca 2+ affinity at the regulatory sites and reduce the Ca 2+ 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 Ca 2+ affinity for two low‐affinity sites. Replacement of endogenous troponin C in skinned muscle fibers by TnC with mutations V45T or M48A increased the Ca 2+ sensitivity of their tension development. These results show that the model can be used to construct mutants that regulate muscle contraction at lower Ca 2+ 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.