Key Points
- To identify novel, physiologically relevant direct interactions between rabbit skeletal muscle troponin C and troponin I using zero-length chemical cross-linking.
- Activated rabbit skeletal muscle troponin C using EDC and NHS, then combined it with equimolar troponin I to form zero-length cross-linked complexes.
- Digested the cross-linked product using cyanogen bromide, trypsin, and Staphylococcus aureus V8 protease.
- Purified the resulting cross-linked peptide fragments using reverse-phase HPLC and identified contact sites via sequence analysis.
- Cross-links formed between the regulatory calcium-binding site II of troponin C (residues 46–78) and segments of troponin I spanning residues 92–167.
- Lys-105 and Lys-107 within the inhibitory region of troponin I were identified as the primary cross-linked residues, demonstrating direct interaction with the amino-terminal domain of troponin C.
Structured PICO
PPopulationRabbit skeletal muscle troponin C (TnC) and troponin I (TnI) complex
IIntervention1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) cross-linking
OOutcomeIdentification of cross-linked residues between TnC and TnIsurrogate
This study provides the first biochemical evidence for a direct interaction between the N-terminal domain of troponin C and the inhibitory region of troponin I.