Tb(III) binding as a probe of Ca(II) binding sites in proteins

ArticleinJournal of the American Chemical Society 98(25):8255-60 · January 1977with15 Reads
Impact Factor: 12.11 · DOI: 10.1021/ja00441a060 · Source: PubMed

Upon addition of Tb 3+ to 40 proteins, many but not all known to interact with Ca 2+, 36 show a characteristic green emission from Tb 3+ upon excitation in the aromatic region. Energy transfer to Tb 3+ is observed from tryptophan in a majority of cases, but transfer from tyrosine and phenylalanine also occurs. Circular polarization of Tb 3+ emission is observable in nine cases including the muscle proteins, carp parvalbumin and troponin-C. All the serine proteases and their zymogens examined display Tb 3+ emission, a result which suggests that they also bind Ca 2+ relatively specifically. The Tb 3+ emission from elastase is partially circularly polarized as well. Weak partially circularly polarized Tb 3+ emission also appears with pronase, α-amylase, and thermolysin. Due to the unexpected high frequency of nearby aromatic chromophores and Tb 3+ added in proteins, Tb 3+ emission promises to be a useful probe for delineating features of protein structure.

    • "Analyses of the sequence of HGSC reveals that one double clamp motif to be present (the residue sequence 34–39 LSRCNS and the sequence 77–82 NDRLSS, both in the N-td, and both in the wild type and the mutant molecule). We hence undertook to study whether the wild type and V42M HGSC display any difference in their Ca 2+ binding properties, by using the Ca 2+ mimic lanthanide trivalent ion probe Tb 3+ , which binds at Ca 2+ binding sites, and displays enhanced luminescence in the visible region of the spectrum, through fluorescence resonance energy transfer [24,25]. AsFigure 5 shows, Tb 3+ binds to both the wild type and the mutant with about the same spectral features, suggesting that both proteins bind Ca 2+ in comparable manner. "
    [Show abstract] [Hide abstract] ABSTRACT: Human γS-crystallin is an important component of the human eye lens nucleus and cortex. The mutation V42M in the molecule causes severe congenital cataract in children. We compare the structure of the mutant protein with that of the wild type in order to understand how structural changes in the mutant relate to the mechanism of opacification. Both proteins were made using conventional cloning and expression procedures. Secondary and tertiary structural features of the proteins were analyzed using spectral methods. Structural stabilities of the proteins were analyzed using chemical and thermal denaturation methods. Self-aggregation was monitored using extrinsic spectral probes. Molecular modeling was used to compare the structural features of the two proteins. While the wild type and mutant have the same secondary structure, molecular modeling and fluorescence analysis suggest the mutant to have a more open tertiary structure, with a larger hydrophobic surface. Experiments using extrinsic probes reveal that the mutant readily self-aggregates, with the suggestion that the aggregates might be similar to amyloidogenic fibrils. Chemical denaturation indicates that while the wild type exhibits the classic two-state transition, V42M goes through an intermediate state, and has a distinctly lower stability than the wild type. The temperature of thermal unfolding of the mutant is also distinctly lower. Further, the mutant readily precipitates and scatters light more easily than the wild type. The replacement of valine in position 42 by the longer and bulkier methionine in human γS-crystallin perturbs the compact β-sheet core packing topology in the N-terminal domain of the molecule, exposes nonpolar residues thereby increasing the surface hydrophobicity and weakens the stability of the protein, thus promoting self-aggregation leading to light scattering particles. This set of changes in the properties of the mutant offers a molecular insight into the mechanism of opacification.
    Full-text · Article · Dec 2012 · PLoS ONE
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    • "Lanthanide elements have extensive application in industries, agriculture, and biomolecular reactions, so it has become very important to understand the behaviour of trivalent lanthanide ions in biological system [1] [2]. The co-ordination chemistry of lanthanide in solution state has become more important with the increase use of lanthanides as probes in the exploration of the structural function of biomolecular reactions [3] [4] [5] [6] specially due to its ability to replace Ca(II) in a specific manner [7] [8] [9] [10]. "
    [Show abstract] [Hide abstract] ABSTRACT: Absorption difference and comparative absorption spectrophotometric studies involving 4f-4f transitions of Nd(III) and different amino acids: DL-valine, DL-alanine, and β-alanine in presence and absence of Ca(II) and Zn(II) in aqueous and different aquated organic solvents have been carried out. Variations in the spectral energy parameters: Slater-Condon (FK) factor, Racah (EK), Lande factor (ξ4f), nephelauxetic ratio (β), bonding (b1/2), percentage covalency (δ) are calculated to explore the mode of interaction of Nd(III) with different amino acids: DL-valine, DL-alanine, and β-alanine. The values of experimentally calculated oscillator strength (P) and computed values of Judd-Ofelt electric dipole intensity parameters, Tλ (λ = 2,4,6), are also determined for different 4f-4f transitions. The variation in the values of P and Tλ parameters explicitly shows the relative sensitivities of the 4f-4f transitions as well as the specific correlation between relative intensities, ligand structures, and nature of Nd(III)-ligand interaction.
    Full-text · Article · Dec 2009 · International Journal of Spectroscopy
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    • "In this paper, lanthanide ions were used as ion probes to study the mechanism of ion-activation of DHFR from chicken liver. Lanthanide ions are ideal since they have similar effective ionic radii and coordination chemistry as that of Ca 2+ (Evants 1990) in addition to the useful spectroscopic and magnetic properties (Brittain et al. 1976; Breen et al. 1985; Mulqueen et al. 1985). Further more, the use of lanthanide ions as calcium probes in the structural studies of calcium binding proteins (Matthews & Weaver 1974; Epstein et al. 1977) has been widely applied. "
    [Show abstract] [Hide abstract] ABSTRACT: The influences of mono-, bi- and trivalent metal ions (as chloride salts) on the activity of dihydrofolate reductase (DHFR) from chicken liver have been studied to elucidate the mechanism of ion-activation of this enzyme. The results show that monovalent ions (Na+ and K+) activate DHFR at low concentration reaching a maximum activation of about 2.5 fold at 0.4-0.5 M and declining at higher concentrations. Ca2+ shows similar activation but at lower concentration, reaching a maximum at 0.1 M; activity declines with further increases in concentration. At very high concentration (> 0.4 M), Ca2+ is inhibitory. The trivalent lanthanide ions, however, show a dramatic inhibition of activity of DHFR even at very low concentration. The activity of DHFR declines to 50% of that of the control at 0.02 mM EuCl3. Intrinsic fluorescence measurements show that the ion-dependent activation in the presence of mono- and bivalent metal ions is due to the conformational changes in the protein. Energy transfer phenomenon suggests that the specific interaction of Eu3+ with Trp24 located in a loop at the active site of DHFR is responsible for the strong inhibition. The possible mechanism for the ion-inhibition is proposed and discussed.
    Preview · Article · Sep 2000 · BioMetals
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