title: Comparison of terbium (III) luminescence enhancement in mutants of EF hand calcium binding proteins.
abstract: The luminescent isomorphous Ca2+ analogue, Tb3+, can be bound in the 12-amino acid metal binding sites of proteins of the EF hand family, and its luminescence can be enhanced by energy transfer from a nearby aromatic amino acid. Tb3+ can be used as a sensitive luminescent probe of the structure and function of these proteins. The effect of changing the molecular environment around Tb3+ on its luminescence was studied using native Cod III parvalbumin and site-directed mutants of both oncomodulin and calmodulin. Titrations of these proteins showed stoichiometries of fill corresponding to the number of Ca2+ binding loops present. Tryptophan in binding loop position 7 best enhanced Tb3+ luminescence in the oncomodulin mutant Y57W, as well as VU-9 (F99W) and VU-32 (T26W) calmodulin. Excitation spectra of Y57F, F102W, Y65W oncomodulin, and Cod III parvalbumin revealed that the principal Tb3+ luminescence donor residues were phenylalanine or tyrosine located in position 7 of a loop, despite the presence of other nearby donors, including tryptophan. Spectra also revealed conformational differences between the Ca2+- and Tb(3+)-bound forms. An alternate binding loop, based on Tb3+ binding to model peptides, was inserted into the CD loop of oncomodulin by cassette mutagenesis. The order of fill of Tb3+ in this protein reversed, with the mutated loop binding Tb3+ first. This indicates a much higher affinity for the consensus-based mutant loop. The mutant loop inserted into oncomodulin had 32 times more Tb3+ luminescence than the identical synthetic peptide, despite having the same donor tryptophan and metal binding ligands. In this paper, a ranking of sensitivity of luminescence of bound Tb3+ is made among this subset of calcium binding proteins. This ranking is interpreted in light of the structural differences affecting Tb3+ luminescence enhancement intensity. The mechanism of energy transfer from an aromatic amino acid to Tb3+ is consistent with a short-range process involving the donor triplet state as described by Dexter (Dexter, D. L. (1953) J. Chem. Phys. 21, 836). This cautions against the use of the Förster equation in approximating distances in these systems.
authors.affiliation: Institute for Biological Sciences, National Research Council of Canada, Ottawa.
authors.forename: C W
authors.initials: CW
authors.lastname: Hogue
authors.forename: J P
authors.initials: JP
authors.lastname: MacManus
authors.forename: D
authors.initials: D
authors.lastname: Banville
authors.forename: A G
authors.initials: AG
authors.lastname: Szabo
mesh_terms: D000595:Amino Acid Sequence; D002135:Calcium-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D002147:Calmodulin / Q000235:genetics* / Q000378:metabolism; D002412:Cations; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D009154:Mutation; D010320:Parvalbumins / Q000235:genetics* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013725:Terbium / Q000737:chemistry*
chemical_list: D002135:Calcium-Binding Proteins; D002147:Calmodulin; D002412:Cations; D010320:Parvalbumins; C034733:oncomodulin; D013725:Terbium