title: Evidence for two-site binding of troponin I inhibitory peptides to the N and C domains of troponin C.
abstract: The interactions of two troponin I peptides, Ip1 (residues 96-116) and Ip2 (residues 104-116), with spectral probe mutants F29W and F105W of intact troponin C (TnC) and of isolated N (residues 1-90) and C (residues 88-162) domains of TnC have been examined. Ip-induced fluorescence emission spectral changes were observed with all four proteins in the presence of Ca2+. Different dependencies of these spectral changes on Ip concentration for intact F29W and F105W are interpreted in terms of two binding sites on TnC. The binding of Ip1 to the C domain (KD1 = 0.50 microM) is 20-40-fold stronger than to the N domain. The binding affinity of Ip1 to both the N and C domains is greater than that of Ip2. The binding strengths of Ip1 to the N domain of intact F29W and isolated F29W/ND are the same within experimental error; that to isolated F105W/CD is weakened by 5-6-fold relative to the C domain of intact F105W. Ip-induced fluorescence changes are dependent on the presence of Ca2+ and are not seen in the presence of Mg2+ alone nor in the absence of divalent cations. This is true even though Ip2 binds to TnC under all three conditions, as demonstrated by affinity chromatography. The accumulated evidence indicates that the F-->W mutations have not significantly affected the binding of Ip peptides to TnC.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Biochemistry, University of Alberta, Edmonton, Canada.
authors.forename: J R
authors.initials: JR
authors.lastname: Pearlstone
authors.forename: L B
authors.initials: LB
authors.lastname: Smillie
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D002846:Chromatography, Affinity; D009154:Mutation; D010455:Peptides / Q000378:metabolism*; D011485:Protein Binding; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D014336:Troponin / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D019209:Troponin C; D019210:Troponin I
chemical_list: D010455:Peptides; D014336:Troponin; D019209:Troponin C; D019210:Troponin I; D002118:Calcium