Photochemical cross-linking between rabbit skeletal troponin subunits. Troponin I-troponin T interactions.

The heterobifunctional photoaffinity probe, AGTC (N-(4-azidobenzoyl-2-3Hglycyl)-S-(2-thiopyridyl) cysteine), was attached to cysteines 48 and 64 of troponin I to determine which component of rabbit skeletal troponin was in close proximity to these cysteines. The reconstituted troponin complex (AGC labeled CM-TnI, TnT, and TnC) was photolyzed and separated using DEAE-Sephadex chromatography in the presence of 8 M urea and absence of reducing agent. Radioactivity measurements indicated that 12% of the cross-linker reacted with solvent and 88% with protein. The percentage of radiolabel found in TnI, TnI-TnT, and TnI-TnC complexes was 35, 55, and 10%, respectively. These results have shown that both TnT and TnC are within 14 A of one or both cysteines 48 and 64 of TnI. Of the total radiolabel found in TnT, 33 and 23% was found in the two cyanogen bromide fragments, CB4 (residues 176-230) and CB2 (residues 71-151). The most likely interpretation of the cross-linking results is that one of the interaction sites between TnI and TnT is an ionic interaction involving the region around cysteines 48 and 64 of TnI (residues 28-82) with the CB5 region of TnT (residues 135-185).