title: 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.
abstract: Contraction of vertebrate striated muscle is regulated by the strong Ca(2+)-dependent interaction between troponin I (TnI) and troponin C (TnC). To critically evaluate this interaction, we generated four recombinant deletion fragments of rabbit fast skeletal TnI: the NH2-terminal fragment (TnI1-94), the NH2 terminus and the inhibitory region (TnI1-120), the inhibitory region and the COOH terminus (TnI96-181), and the COOH-terminal fragment (TnI122-181) containing amino acid residues 1-94, 1-120, 96-181, and 122-181, respectively. Native TnC and seven thiol mutants, containing single cysteine residues in the two globular 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-maleimidobenzophenone, and their interaction with the recombinant TnI fragments and the synthetic inhibitory peptide (TnI98-114, 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 accessible to both of the globular domains of TnC, (b) indicated that linkage of the NH2- and COOH-terminal sequences to the inhibitory region of TnI (TnIir) caused marked enhancement of cross-linking with native TnC and all seven thiol mutants, and (c) identified the region in TnC where TnIir binds as that containing residues 98, 133, 158, and 57. Thus, the results suggest that TnI and TnC may adopt flexible and dynamic conformations in which multiple interactions involving various domains of the two polypeptides occur and TnIir acting as a linker facilitates 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 functional region of TnI. Whereas the NH2-terminal region of TnI stabilizes binding to TnC and TnT, the COOH-terminal region stabilizes TnC and actin binding.
authors.affiliation: Department of Anatomy and Cellular Biology, Tufts University, Boston, Massachusetrs 02111, USA.
authors.forename: P K
authors.initials: PK
authors.lastname: Jha
authors.forename: C
authors.initials: C
authors.lastname: Mao
authors.forename: S
authors.initials: S
authors.lastname: Sarkar
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003432:Cross-Linking Reagents; D003545:Cysteine / Q000378:metabolism; D008301:Maleimides; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism; D015879:Myosin Subfragments / Q000378:metabolism; D009218:Myosins / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism / Q000494:pharmacology; D010782:Photolysis; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011817:Rabbits; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion; D013439:Sulfhydryl Reagents; D014336:Troponin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019209:Troponin C; D019210:Troponin I
chemical_list: D003432:Cross-Linking Reagents; D008301:Maleimides; D015879:Myosin Subfragments; D010446:Peptide Fragments; D011994:Recombinant Proteins; D013439:Sulfhydryl Reagents; D014336:Troponin; D019209:Troponin C; D019210:Troponin I; C000245:N-(4-(2-benzimidazolyl)phenyl)maleimide; D009218:Myosins; D003545:Cysteine