Cross-linking of rabbit skeletal muscle troponin subunits: labeling of cysteine-98 of troponin C with 4-maleimidobenzophenone and analysis of products formed in the binary complex with troponin T and the ternary complex with troponins I and T

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Abstract
The sulfhydryl-specific, heterobifunctional, photoactivatable cross-linker 4-maleimidobenzophenone (BPMal) was used to study the interaction of rabbit skeletal muscle troponin subunits TnC, TnT, and TnI. TnC was labeled at Cys-98 by the maleimide moiety of BPMal and then mixed with either TnT alone or TnI plus TnT, in the presence of Ca2+. Upon photolysis TnI and/or TnT formed covalent cross-links with TnC. The cross-linked TnC-TnT heterodimer obtained from the binary complex was digested into progressively smaller cross-linked peptides that were purified by HPLC and then characterized by amino acid analysis and sequencing. An initial cross-linked CBNr fraction contained the expected peptide CB9 (residues 84-135) of TnC, plus CNBr peptides spanning residues 152-230 of TnT. Results from a peptic digest of CNBr cross-linked fraction permitted the identification of residues 159-197 as the most highly cross-linked region in TnT. A final subtilism digest yielded a heterogeneous cross-linked fraction, which suggested an especially high degree of cross-links was formed in the vicinity of residues 175-178 (Met-Lys-Lys-Lys) of TnT. Although this region of TnT had previously been implicated in binding, we show here for the first time that it is close to Cys-98 of TnC. In an analogous study on the binary complex of TnC and TnI [Leszyk, J., Collins, J.H., Leavis, P.C., and Tao, T. (1987) Biochemistry 26, 7042-7047], we previously showed that Cys-98 of TnC was cross-linked mainly to CN4, the "inhibitory region", of TnI. In this report we analyze the cross-linked products from the complex of all three troponin subunits and find that CN4 (residues 96-116) of TnI and CB4 (residues 176-230) of TnT are the predominant cross-linked CNBr peptides in these two proteins. This indicates that the Cys-98 region of TnC interacts with TnI and TnT in a similar manner in both the ternary and binary complexes.