Thermodynamics of cation binding to Nereis sarcoplasmic calcium-binding protein. Direct binding studies, microcalorimetry and conformational changes.
Nereis sarcoplasmic calcium-binding protein contains three functional EF-hand sites which bind Ca2+ or Mg2+ competitively. Here it was confirmed over a large range of [Mg2+] that the positive cooperativity in binding of Ca2+ (nH = 2) is the result of allostery in Mg2+ dissociation. At pH 7.5, Ca2+ or Mg2+ binding provokes the release of 1.4 mol proton/mol protein, whereas no protons are released during Ca(2+)-Mg2+ exchange. The enthalpy change as a function of Ca2+ binding yields a two-step curve with an inflection point at 1 mol Ca2+/mol protein and a maximum of -66 kJ/mol at 3 mol Ca2+/mol protein. Binding of three Mg2+ ions is cooperative (nH = 1.8) with a maximal enthalpy change of -15.1 kJ/mol protein. Difference spectroscopy led to the conclusion that, in the metal-free protein, the structure around the aromatic residues is well organized, but that Tyr and Trp residues are still solvent-exposed. Upon Ca2+ binding Tyr and Trp spectra are blue-shifted, but some Trp residues are confined to a positively charged pocket. Examination of the Ca(2+)-saturated three-dimensional crystal structure confirmed that Trp4 and Trp57 are located in such pockets or clefts, close to the surface. During the allosteric T----R transition, promoted by binding of the first Mg2+, the Trp residues move to a hydrophobic environment. For both Ca2+ and Mg2+, the enthalpy change and the conformational change in the environment of the aromatic residues is much more pronounced in the first, than in the subsequent two binding steps. In this respect, the latter seem to be equivalent.
Luan-Rilliet
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Y
Department of Biochemistry, University of Geneva, Switzerland.
Milos
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M
Cox
J A
JA
D000494:Allosteric Regulation; D000495:Allosteric Site; D000818:Animals; D000845:Annelida / Q000378:metabolism*; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002151:Calorimetry; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism*; D008962:Models, Theoretical; D011487:Protein Conformation; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*
D002135:Calcium-Binding Proteins; D008274:Magnesium; D002118:Calcium