A new heterobifunctional cross-linking reagent for the study of biological interactions between proteins. II. Application to the troponin C-troponin I interaction.

A simple chromatographic procedure using DEAE-Sephadex has been established to isolate the troponin I-troponin C complex from unbound troponin I (TnI) and troponin C (TnC). A 1:1 complex can be formed between bovine cardiac carboxamidomethylated troponin I and rabbit skeletal troponin C. The formation of the complex is calcium dependent. It is stable to DEAE-chromatography in 6 M urea, 3 mM Ca2+ and can be dissociated on DEAE-chromatography in the presence of 6 M urea, 1 mM ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. TnC was modified with the photoaffinity probe AGTC (N-(4-azidobenzoyl-[2-3H]glycyl)-S-(2-thiopyridyl)-cysteine) at its single cysteine residue (position 98). Photolysis of the CM (carboxamidomethylated)-TnI-AGC-TnC complex resulted in the formation of a covalently linked 1:1 complex. The isolated covalently linked complex could be treated with dithiothreitol to reduce the disulfide bond between N-(4-azidobenzoyl-[2-3H]glycyl)-cysteine (AGC) and TnC to complete the transfer of the radiolabeled AGC from cysteine 98 on TnC to CM-TnI. The CM-TnI-AGC was isolated from TnC on DEAE-chromatography in 6 M urea, 1 mM EGTA, 1 mM dithiothreitol buffer. The formation of the covalent bond between the photoaffinity probe and TnI indicates the close proximity of TnI to cysteine 98 on the TnC. These results demonstrate the general utility of the new heterobifunctional cross-linking reagent to study protein interactions.