Key Points
- To sequence-specifically assign the methionine methyl resonances in recombinant cardiac troponin C and evaluate their structural responses to calcium binding across distinct domains.
- Metabolically labeled 10 methionine methyl groups in recombinant cardiac troponin C with carbon-13 ([13C-methyl]Met).
- Assigned epsilon-carbon and epsilon-hydrogen chemical shifts using two-dimensional heteronuclear single- and multiple-quantum coherence (HSMQC) spectroscopy paired with systematic Met-to-Leu site-directed mutagenesis.
- Tracked chemical shift alterations upon calcium titration at high-affinity C-terminal (sites III and IV) and low-affinity N-terminal (site II) binding sites.
- All 10 methionine methyl resonances were assigned sequence-specifically, with negative functional impacts appearing only when both Met 45 and Met 81 were mutated to leucine.
- Calcium binding at high-affinity C-terminal sites III and IV caused large chemical shift changes in C-terminal methionines alongside minor shifts in N-terminal Met 47 and Met 81.
- Calcium binding to low-affinity N-terminal site II induced substantial shift changes specifically in Met 45, Met 80, and Met 81 without affecting C-terminal residues, demonstrating distinct domain-specific structural shifts.
Structured PICO
PPopulationRecombinant cardiac troponin C (cTnC)
IInterventionBinding of Ca2+ to high affinity C-terminal sites III and IV and low affinity N-terminal site II
OOutcomeChanges in epsilon H and epsilon C chemical shifts of Met residuessurrogate
Assigned methyl Met chemical shifts in cardiac troponin C can serve as structural markers to study conformational transitions upon calcium binding.