title: Kinetics of conformational changes in Nereis sarcoplasmic calcium-binding protein upon binding of divalent ions.
abstract: The sarcoplasmic calcium-binding protein (SCP) of the sandworm Nereis possesses three Ca2(+)-Mg2+ sites but no Ca2(+)-specific site. Binding of Mg2+, but not of Ca2+, displays a marked positive cooperativity. The apparent cooperativity of Ca2+ binding in the presence of Mg2+ results from the allostery in Mg2+ dissociation. Binding of the first Ca2+ or Mg2+ induces all the conformational change, monitored by Trp fluorescence. In displacement reactions the conformational changes occur in the step SCP.Mg3----SCP.Ca1Mg2. Stopped-flow experiments indicate that Trp fluorescence changes upon Ca2(+)-binding are instantaneous whereas Mg2(+)-binding involves a fast pre-equilibrium (Keq = 28 M-1), followed by two slow consecutive conformational changes with k1 = 13.5 s-1 and k2 = 0.21 s-1. The fluorescence change after dissociation of Ca2+ from SCP is monophasic with k = 0.02 s-1; that after Mg2+ dissociation is biphasic with k1 = 0.8 s-1 and k2 = 0.1 s-1. Trp life time measurements also indicate that Ca2(+)- and Mg2(+)-induced conformational changes are completely different. Displacement of bound Ca2+ by Mg2+ can be described by two consecutive reactions in which the first (without fluorescence change) corresponds to the dissociation of the last Ca2+ (k1 = 2.4 s-1) and the second (k2 = 0.45 s-1) to the final conformational change observed upon direct Mg2+ binding. Displacement of bound Mg2+ by Ca2+ follows the kinetic scheme of simple competition; the conformational rate constant approaches asymptotically (up to the limit of 129 s-1) the dissociation rate of Mg2+ as the concentration of Ca2+ increases. In summary, after fast dissociation of Ca2+ or Mg2+, Nereis SCP slowly converts to the metal-free configuration, but in Ca2(+)-Mg2+ exchange reactions, the conformational changes are nearly as fast as the cation dissociation reactions.
authors.affiliation: Laboratory of Chemical and Biological Dynamics, University of Leuven, Belgium.
authors.forename: Y
authors.initials: Y
authors.lastname: Engelborghs
authors.forename: K
authors.initials: K
authors.lastname: Mertens
authors.forename: K
authors.initials: K
authors.lastname: Willaert
authors.forename: Y
authors.initials: Y
authors.lastname: Luan-Rilliet
authors.forename: J A
authors.initials: JA
authors.lastname: Cox
mesh_terms: D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002413:Cations, Divalent; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism / Q000494:pharmacology; D008433:Mathematics; D008962:Models, Theoretical; D011077:Polychaeta / Q000378:metabolism*; D011487:Protein Conformation; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
chemical_list: D002135:Calcium-Binding Proteins; D002413:Cations, Divalent; D008274:Magnesium; D002118:Calcium