Lanthanide ion luminescence probes. Characterization of metal ion binding sites and intermetal energy transfer distance measurements in calcium-binding proteins. 1. Parvalbumin

Abstract

Eu(III) laser excitation spectroscopy of the 7F0 leads to 5D0 transition reveals spectral features characteristic of the occupation of the CD and EF Ca(II)-binding sites of parvalbumin by Eu(III) ions. In addition, at pH 6.5, a signal attributable to binding at a third site is observed. This feature is abolished upon lowering the pH to 3.8. At pH 6.5, the appearance of this feature correlates with the decrease in luminescence intensity during titrations of parvalbumin with Eu(III) or Tb(III) after more than approximately 1.8 equiv of either of these ions has been added. Eu(III) ions in the primary sites coordinate zero to two H2O molecules while the third site involves about three coordinated H2O molecules. Parvalbumins in which mixed pairs of lanthanide ions, Ln(III), occupy the CD and EF sites were prepared. Nonradiative energy transfer between Eu(III) and Tb(III) acting as luminescent donors and various other Ln(III) ions serving as acceptors was observed by monitoring the excited-state lifetimes of the donor ions using a pulsed dye laser apparatus. With the assumption of a Förster-type dipole--dipole mechanism, inter-binding-site distance estimates were made from our measurements and are in reasonable agreement with the distance (11.8 A) obtained by X-ray crystallography, especially when Eu(III) is the donor. R0 values (critical distances for 50% energy transfer) in H2O solution range from 9.2 A for the Tb(III)--Ho(III) donor-acceptor pair down to 5.7 A for the Eu(III)--Ho(III) pair.

Fluorescence energy transfer measurement of distances between ligand binding sites of tubulin and its implication for protein‐protein interaction

“…complexes may make K' close to 2/3 (Rhee et al, 1981). Thus, a value of 33 A, between the DCVJ and ruthenium red sites may be accepted with a high degree of confidence.…”

Luminescence Resonance Energy Transfer in Heterodinuclear LnIII Complexes for Sensing Biologically Relevant Anions

“…[21,30,[42][43][44][45] In addition, LRET has been applied to mapping metal ion binding sites of biomolecules. [46] In particular, Ln III are good Ca II surrogates and have been used to study multisite calcium binding proteins such as Troponin-C, [47] parvalbumin, [48,49] and thermolysin. [50] In these studies, mixed lanthanide cations were bound to the proteins and used to estimate distances between metal ion binding sites.…”

“…[51] The Eu III and Nd III donor-acceptor pair is particularly useful. [48,50,51] Nd III has one of the largest extinction coefficients (~10 m -1 cm -1 ) of all the Ln III , which facilitates LRET over longer distances. [52] Eu III is useful in these photoluminescent experiments because the ion has an excitation transition ( [53][54][55][56][57] Chrysochoos and coworkers studied Tb III and Eu III donor and acceptor LRET pairs in DMSO [58,59] and aqueous solutions.…”

Identification of Metal-binding Sites in Rat Brain Calcium-binding Protein

“…Energy transfer experiments from Trp residues to metal atoms are useful in the determination of Trp to metal distances in proteins (14,17,29,30). The intensity of the terbium emission excited by energy transfer depends on the distance between and the quantum yield of the donor and acceptor, as well as several other parameters (13).…”