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.
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 CNBr 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 the CNBr cross-linked fraction permitted the identification of residues 159-197 as the most highly cross-linked region in TnT. A final subtilisin digest yielded a heterogeneous cross-linked fraction, which suggested that 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., & 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Leszyk
J
J
Department of Biological Chemistry, School of Medicine, University of Maryland, Baltimore 21201.
Collins
J H
JH
Leavis
P C
PC
Tao
T
T
D000818:Animals; D001577:Benzophenones / Q000494:pharmacology; D003432:Cross-Linking Reagents; D003545:Cysteine; D066298:In Vitro Techniques; D008301:Maleimides; D009132:Muscles / Q000378:metabolism; D011487:Protein Conformation / Q000187:drug effects; D011817:Rabbits; D014336:Troponin / Q000378:metabolism*; D019209:Troponin C; D019210:Troponin I; D020107:Troponin T
D001577:Benzophenones; D003432:Cross-Linking Reagents; D008301:Maleimides; D014336:Troponin; D019209:Troponin C; D019210:Troponin I; D020107:Troponin T; C046109:benzophenone-4-maleimide; D003545:Cysteine