title: Stoichiometry of calcium binding to a synthetic heterodimeric troponin-C domain.
abstract: In this work we describe calcium binding to two synthetic 34-residue peptides, determined by 1H-nmr spectroscopy. The peptides investigated, SCIII and SCIV, encompass the calcium-binding sites III and IV, respectively, of troponin-C. In the absence of calcium it has previously been shown that each of these peptides possesses little regular secondary structure. Further, the 1H-nmr spectra of an equimolar mixture of both of these apo-peptides (apo-SCIII/SCIV) shows that little interaction occurs between peptides. Upon calcium binding the spectral changes that occur to SCIII/SCIV are consistent with global conformational changes in both peptides. We have shown previously that these conformational changes are a product of calcium binding to SCIII and SCIV to form a two-site heterodimer Ca2-SCIII/SCIV. It is proposed that this calcium-induced folding proceeds via calcium binding to SCIII to form Ca-SCIII, peptide association with apo-SCIV to form the heterodimer Ca-SCIII/SCIV, and calcium binding to form Ca2-SCIII/SCIV. The dissociation constants involved in this pathway, K1, Kd, and K2, respectively, have been determined by stoichiometric calcium titration of SCIII/SCIV, monitored by 1H-nmr spectroscopy. Using this procedure it has been determined that K1 = 3 microM, Kd = 10 microM, and K2 = 2 microM.
authors.affiliation: Department of Biochemistry, University of Alberta, Edmonton, Canada.
authors.forename: G S
authors.initials: GS
authors.lastname: Shaw
authors.forename: R S
authors.initials: RS
authors.lastname: Hodges
authors.forename: B D
authors.initials: BD
authors.lastname: Sykes
mesh_terms: D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D012996:Solutions; D014336:Troponin / Q000737:chemistry / Q000378:metabolism*; D019209:Troponin C
chemical_list: D002135:Calcium-Binding Proteins; D010446:Peptide Fragments; D012996:Solutions; D014336:Troponin; D019209:Troponin C; D002118:Calcium