HD exchange and PLIMSTEX determine the affinities and order of binding of Ca²⁺ with troponin C

Troponin C (TnC), present in all striated muscle, is the Ca ²⁺-activated trigger that initiates myocyte contraction. The binding of Ca²⁺ to TnC initiates a cascade of conformational changes involving the constituent proteins of the thin filament. The functional properties of TnC and its ability to bind Ca²⁺ have significant regulatory influence on the contractile reaction of muscle. Changes in TnC may also correlate with cardiac and various other muscle-related diseases. We report here the implementation of the PLIMSTEX strategy (protein ligand interaction by mass spectrometry, titration, and H/D exchange) to elucidate the binding affinity of TnC with Ca²⁺ and, more importantly, to determine the order of Ca²⁺ binding of the four EF hands of the protein. The four equilibrium constants, K₁ = (5 ± 5) x 10⁷ M ^-1, K₂ = (1.8 ± 0.8) x 10⁷ M ^-1, K₃ = (4.2 ± 0.9) x 10⁶ M ^-1, and K₄ = (1.6 ± 0.6) x 10⁶ M ^-1, agree well with determinations by other methods and serve to increase our confidence in the PLIMSTEX approach. We determined the order of binding to the four EF hands to be III, IV, II, and I by extracting from the H/DX results the deuterium patterns for each EF hand for each state of the protein (apo through fully Ca²⁺ bound). This approach, demonstrated for the first time, may be general for determining binding orders of metal ions and other ligands to proteins.