Characterization of the lanthanide ion-binding properties of calcineurin-B using laser-induced luminescence spectroscopy.
Calcineurin (CaN) is a Ca2+/calmodulin-dependent protein phosphatase found in brain and other tissues. It is a heterodimer consisting of a catalytic subunit (CaN-A) and a Ca(2+)-binding regulatory subunit (CaN-B). The primary structure of CaN-B indicates that it, like calmodulin, is an EF-hand protein and binds four Ca2+ ions. Eu3+, due to its favorable spectroscopic and chemical properties, has been substituted for Ca2+ in CaN-B to determine the metal ion-binding properties of this "calmodulin-like" protein. Excitation of the 7F0-->5D0 transition of Eu3+ results in a spectrum similar to that of calmodulin, consisting of three peaks. Analysis of the spectral titration curves reveals four Eu(3+)-binding sites in CaN-B. The affinities vary: sites I and II have dissociation constants of 1.0 +/- 0.2 and 1.6 +/- 0.4 microM, respectively; the values for sites III and IV are Kd = 140 +/- 20 and Kd = 20 +/- 10 nM, respectively. Binding of Tb3+ is slightly weaker. Tb3+ luminescence, sensitized by tyrosine, reveals that for lanthanides the highest affinity sites lie in the C-terminal domain. Energy transfer distance measurements between Eu3+ and Nd3+ in sites III and IV reveal a separation of 10.5 +/- 0.5 A, which suggests that these sites are arranged in a typical EF-hand pair. This information indicates that the overall structure of CaN-B is similar to the dumbbell-shaped proteins troponin-C and calmodulin, but is more like TnC in its metal-binding properties.
Burroughs
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SE
Department of Chemistry, Pennsylvania State University, University Park 16802.
Horrocks
W D
WD
Ren
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H
Klee
C B
CB
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019703:Calcineurin; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002147:Calmodulin / Q000235:genetics; D002417:Cattle; D003001:Cloning, Molecular; D004735:Energy Transfer; D006801:Humans; D007700:Kinetics; D007834:Lasers; D008163:Luminescent Measurements; D008674:Metals, Rare Earth / Q000378:metabolism*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D011485:Protein Binding; D011817:Rabbits; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry / Q000379:methods; D014336:Troponin / Q000235:genetics; D019209:Troponin C
D002135:Calcium-Binding Proteins; D002147:Calmodulin; D008674:Metals, Rare Earth; D014336:Troponin; D019209:Troponin C; D019703:Calcineurin