Key Points
- To test whether replacing hydrophobic residues with polar residues in the regulatory domain of troponin C increases calcium affinity and reduces the calcium threshold required for muscle activation.
- Engineered two troponin C point mutants (V45T and M48A) guided by crystallographic models of calcium-induced conformational transitions.
- Conducted direct calcium-binding assays to evaluate affinity changes at regulatory sites I and II.
- Substituted endogenous troponin C in skinned muscle fibers with the mutant proteins to measure calcium sensitivity during isometric tension development.
- Both V45T and M48A mutations increased calcium-binding affinity at the two low-affinity regulatory sites compared to wild-type troponin C.
- Incorporation of V45T or M48A troponin C into skinned muscle fibers heightened calcium sensitivity, producing isometric tension development at lower calcium concentrations.
Structured PICO
PPopulationSkinned muscle fibers and troponin-C mutants
IInterventionTroponin-C mutants (V45T and M48A)
CComparatorEndogenous troponin C
OOutcomeCalcium affinity and calcium sensitivity of tension developmentsurrogate
Troponin-C mutants V45T and M48A increase calcium affinity and sensitivity of muscle tension development, supporting the proposed calcium-induced conformational change model.