CHARACTERIZATION OF THE LANTHANIDE ION-BINDING PROPERTIES OF CALCINEURIN-B USING LASER-INDUCED LUMINESCENCE SPECTROSCOPY

Calcineurin (CaN) is a Ca2+/calmodulin-dependent protein phosphatase f ound in brain a nd other tissues. It is a heterodimer consisting of a catalytic subunit (CaN-A) and a Ca2+-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 favor able spectroscopic and chemical properties, has been substituted for C a2+ in CaN-B to determine the metal ion-binding properties of this ''c almodulin-like'' protein. Excitation of the F-7(0)-->D-5(0) transition of Eu3+ results in a spectrum similar to that of calmodulin, consisti ng of three peaks. Analysis of the spectral titration curves reveals f our Eu3+-binding sites in CaN-B. The affinities vary: sites I and II h ave dissociation constants of 1.0 +/- 0.2 and 1.6 +/- 0.4 mu M, respec tively; the values for sites III and IV are K-d = 140 +/- 20 and K-d = 20 +/- 10 nM, respectively. Binding of Tb3+ is slightly weaker. Tb3luminescence, 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 Angstrom, which suggests that these site s are arranged in a typical EF-hand pair. This information indicates t hat the overall structure of CaN-B is similar to the dumbbell-shaped p roteins troponin-C and calmodulin, but is more like TnC in its metal-b inding properties.