A RECOMBINANT MONOCYSTEINE MUTANT (SER TO CYS-155) OF FAST SKELETAL TROPONIN-T - IDENTIFICATION BY CROSS-LINKING OF A DOMAIN INVOLVED IN A PHYSIOLOGICALLY RELEVANT INTERACTION WITH TROPONIN-C AND TROPONIN-I

Troponin T (TnT), a subunit of the heterotrimeric troponin (Tn) comple x, is essential for the Ca2+ regulation of vertebrate striated muscle contraction both in vivo and in vitro. With the exception of bovine ca rdiac TnT, all known vertebrate TnT isoforms lack a thiol group, a pro perty which makes the wild-type proteins unsuitable as cross-linking s ubstrate. We generated a mutant human fast skeletal TnT in which Ser(1 55) was changed to Cys (TnT-Cys(155)). Mutation of this residue in TnT as well as in vitro expression in Escherichia coli and purification o f the recombinant mutant protein did not affect its biological propert ies in terms of in vitro binding to troponin I (TnI), troponin C (TnC) , actin-tropomyosin (actin-Tm), and actomyosin ATPase activity. TnT-Cy s(155) was labeled with 4-maleimidobenzophenone (BP-TnT(155)) and phot o-cross-linked to TnI, TnC, Tm, and all of the thin filament proteins. BP-TnT(155) did not cross-link to Tm and showed weak Ca2+/Mg2+-indepe ndent cross-linking with TnI in the binary complex and in the presence of all thin filament protein components. BP-TnT(155) showed Ca2+/Mg2-dependent cross-linking with TnC in the binary and ternary complexes and Ca2+-favored cross-linking with TnI in the ternary complex. Thus, residue 155 of TnT is within 10 Angstrom (the length of cross-linker) of TnC in the presence or absence of Ca2+ and comes within 10 Angstrom of both TnI and TnC in the presence of Ca2+. TnT residue 155 is in cl ose proximity to or may even partly encompass the Tm binding site. The se results suggest that TnT, in association with TnI, may participate in the ''information transfer'' mediated by the Ca2+ binding signal fr om TnC to Tm and the region around TnT residue 155 probably acts as a linker between troponin and actin-Tm in this signal transmission proce ss. Our results also suggest that TnT contains at least one Ca2+/Mg2+- dependent TnC binding region located between its Tm and TnI binding re gions. A recombinant truncated fragment of TnI, TnI(96-181), containin g amino acid residues 96-181 and labeled with BP at Cys-133, failed to cross-link with TnT, indicating that the region around Cys-133 of TnI is not involved in binary interaction with TnT.