Characterization of Eu(III) binding to a series of calmodulin binding site mutants using laser-induced Eu(III) luminescence spectroscopy.
Laser-induced luminescence techniques were used in a rigorous evaluation of the Eu(3+)-binding behavior of a recombinant (Drosophila melanogaster) calmodulin and a series of calmodulin binding site mutants in which the bidentate glutamic acid residue in position 12 of each metal ion binding loop is systematically replaced with lysine. For the range of Ca2+ concentrations at which calmodulin functions (10(-5)-10(-6) M), Ca2+ binding is effectively eliminated at the mutated site; however, the luminescence studies show that the Eu3+ ion binds to the modified site with reduced affinity. The mutations do not significantly change the intermetal ion distances from their wild type values. These were determined by Eu3+-->Nd3+ Förster-type non-radiative energy transfer experiments. Consistent with the results of Ca(2+)-binding studies, mutation of sites II and IV in the N- and C-terminal domains, respectively, produces a larger alteration in the Eu(3+)-luminescence and Eu(3+)-binding behavior than does mutation of sites I and III. Modification of either of the sites in the C-terminus (III or IV, numbered from the amino terminus) causes two additional H2O molecules (four H2O molecules total) to bind to the Eu3+ ion in order to compensate for the loss of the bidentate glutamic acid residue. Consequently, the partner site in the domain loses an H2O molecule, thereby coordinating another ligand from the protein. Mutation of either of the high-affinity Ln(3+)-binding sites (I or II) has global effects on the Eu(3+)-binding behavior of the protein molecule.
Bruno
J
J
Department of Chemistry, Pennsylvania State University 152 Davey Laboratory, University Park 16802, USA.
Horrocks
W D
WD
Beckingham
K
K
D000818:Animals; D001665:Binding Sites / Q000235:genetics*; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000235:genetics* / Q000378:metabolism*; D055598:Chemical Phenomena; D002621:Chemistry; D004331:Drosophila melanogaster; D005063:Europium / Q000378:metabolism*; D007834:Lasers; D008163:Luminescent Measurements; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013057:Spectrum Analysis; D014867:Water / Q000378:metabolism
D002147:Calmodulin; D011994:Recombinant Proteins; D014867:Water; D005063:Europium; D002118:Calcium