Interaction of troponin C and troponin C fragments with troponin I and the troponin I inhibitory peptide.
We have quantitated the interactions of two rabbit skeletal troponin C fragments with troponin I and the troponin I inhibitory peptide. The calcium binding properties of the fragments and the ability of the fragments to exert control in the regulated actomyosin ATPase assay have also been studied. The N- and C-terminal divalent metal binding domains of rabbit skeletal troponin C, residues 1-97 and residues 98-159, respectively, were prepared by specific cleavage at cysteine-98 and separation by gel exclusion chromatography. Both of the troponin C fragments bind calcium. The calcium affinity of the weak sites within the N-terminal fragment is about an order of magnitude greater than is reported for these sites in troponin C, suggesting interaction between the calcium-saturated strong sites and the weak sites. Stoichiometric binding (1:1) of the troponin I inhibitory peptide to each fragment and to troponin C increased the calcium affinities of the fragments and troponin C. Complex formation was detected by fluorescence quenching or enhancement using dansyl-labeled troponin C (and fragments) or tryptophan-labeled troponin I inhibitory peptide. The troponin C fragments bind to troponin I with 1:1 stoichiometry and approximately equal affinities (1.6 x 10(6) M-1) which are decreased 4-fold in the presence of magnesium versus calcium. These calcium effects are much smaller than is observed for troponin C. The summed free energies for the binding of the troponin C fragments to troponin I are much larger than the free energy of binding troponin C. This suggests a large positive interaction free energy for troponin C binding to troponin I relative to the fragments.(ABSTRACT TRUNCATED AT 250 WORDS)
Swenson
C A
CA
Department of Biochemistry, University of Iowa, Iowa City 52242.
Fredricksen
R S
RS
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002850:Chromatography, Gel; D003619:Dansyl Compounds; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D009132:Muscles / Q000737:chemistry*; D010446:Peptide Fragments / Q000378:metabolism*; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014336:Troponin / Q000378:metabolism*; D019209:Troponin C; D019210:Troponin I
D003619:Dansyl Compounds; D005456:Fluorescent Dyes; D010446:Peptide Fragments; D014336:Troponin; D019209:Troponin C; D019210:Troponin I; D008274:Magnesium; D002118:Calcium