PHOTO-CROSS-LINKING OF RABBIT SKELETAL TROPONIN-I DELETION MUTANTS WITH TROPONIN-C AND ITS THIOL MUTANTS - THE INHIBITORY REGION ENHANCES BINDING OF TROPONIN-I FRAGMENTS TO TROPONIN-C

Contraction of vertebrate striated muscle is regulated by the strong C a2+-dependent interaction between troponin I(TnI) and troponin C (TnC) . To critically evaluate this interaction, we generated four recombina nt deletion fragments of rabbit fast skeletal TnI: the NH2-terminal fr agment (TnI(1-94)), the NH2 terminus and the inhibitory region (TnI(1- 120)), the inhibitory region and the COOH terminus (TnI(96-181)), and the COOH-terminal fragment (TnI(122-181)) containing amino acid residu es 1-94, 1-120, 96-181, and 122-181, respectively. Native TnC and seve n thiol mutants, containing single cysteine residues in the two globul ar domains and in the central helix of TnC, e.g., Cys-12, Cys-21, Cys- 57, Cys-89, Cys-122, Cys-133, and Cys-158, were labeled with 4-maleimi dobenzophenone, and their interaction with the recombinant TnI fragmen ts and the synthetic inhibitory peptide (TnI(98-114), residues 98-114) was studied by photo-cross-linking. Extensive cross-linking occurred between various domains of TnC and TnI. The cross-linking patterns (a) showed that both NH2- and COOH-terminal fragments of TnI are accessib le to both of the globular domains of TnC, (b) indicated that linkage of the NH2- and COOH-terminal sequences to the inhibitory region of Tn I (TnI(ir)) caused marked enhancement of cross-linking with native TnC and all seven thiol mutants, and (c) identified the region in TnC whe re TnI(ir) binds as that containing residues 98, 133, 158, and 57. Thu s, the results suggest that TnI and TnC may adopt flexible and dynamic conformations in which multiple interactions involving various domain s of the two polypeptides occur and TnI(ir) acting as a linker facilit ates these interactions. The interaction of TnI and its fragments with actin, TnC, and TnT, considered together with the biological activity indicates that residues 96-120 represent a key structural and functio nal region of TnI. Whereas the NH2-terminal region of TnI stabilizes b inding to TnC and TnT, the COOH-terminal region stabilizes TnC and act in binding.