Weak binding of divalent cations to plasma gelsolin

Calcium binding of swine plasma gelsolin was examined. When applied to ion-exchange chromatography, its elution volume was drastically altered depending on the free Ca2+ concentration of the medium. The presence of two classes of Ca2+ binding sites, high-affinity sites (Kd = 7 microM) and low-affinity sites (Kd = 1 mM), was suggested from the concentration dependence of the elution volume. The tight binding sites were specific for Ca2+. The weakly bound Ca2+ could be replaced by Mg2+ once the tight binding sites were occupied with Ca2+. The binding of metal ions was totally reversible. Circular dichroism measurement of plasma gelsolin indicated that most change in secondary structure was associated with Ca2+ binding to the high-affinity sites. Binding of Mg2+ to the low-affinity sites caused a secondary structural change different from that caused by Ca2+ bound to the high-affinity sites. Gel permeation chromatography exhibited a small change in Stokes radius with and without Ca2+. Microheterogeneity revealed by isoelectric focusing did not relate to the presence of two classes of Ca2+ binding sites. These results indicated that plasma gelsolin drastically altered its surface charge property due to binding of Ca2+ or Ca2+, Mg2+ with a concomitant conformational change.     

Luminescence studies of Tb3+ bound to the high affinity sites of the Ca2+-ATPase of sarcoplasmic reticulum

Direct excitation of lanthanide luminescence with a pulsed dye laser has been used to probe the molecular environment of the high affinity sites of the sarcoplasmic reticulum Ca2+-ATPase. The direct excitation spectrum of Tb3+ bound to these sites has been determined and a luminescence lifetime of approximately 1 ms measured. Measurements of the difference in lifetime of the Tb X ATPase complex in H2O and D2O indicate that there are approximately 2 H2O molecules in the first coordination sphere of Tb3+ bound at the high affinity sites of the ATPase. The results are compared with the properties of Tb3+ binding to high affinity sites of other Ca2+ binding proteins. The binding constant of Tb3+ to the ATPase is in the range of 0.3-5.0 X 10(8) M-1 as inferred from the KI for inhibition of ATP hydrolysis, in agreement with a previous report (Highsmith, S. R., and Head, M. R. (1983) J. Biol. Chem. 258, 6858-6862). The values of the Ca2+ binding constant (approximately 2 X 10(6) M-1) and the cooperative nature (n = 1.9) of Ca2+ protection of Tb3+ inhibition indicate that Tb3+ and Ca2+ compete for the high affinity sites of the ATPase. The results demonstrate that directly-excited Tb3+ luminescence provides unique information on the environment of the Ca2+ binding-transport sites of the SR ATPase.     

Kinetic studies show that Ca2+ and Tb3+ have different binding preferences toward the four Ca2+-binding sites of calmodulin

The stepwise addition of Tb3+ to calmodulin yields a large tyrosine-sensitized Tb3+ luminescence enhancement as the third and fourth ions bind to the protein [Wang, C.-L. A., Aquaron, R. R., Leavis, P. C., & Gergely, J. (1982) Eur. J. Biochem. 124, 7-12]. Since the only tyrosine residues in calmodulin are located within binding sites III and IV, these results suggest that Tb3+ binds first to sites I and II. Recent NMR studies have provided evidence that Ca2+, on the other hand, binds preferentially to sites III and IV. Kinetic studies using a stopped-flow apparatus also show that the preferential binding of Ca2+ and lanthanide ions is different. Upon rapid mixing of 2Ca-calmodulin with two Tb3+ ions, there was a small and rapid tyrosine fluorescence change, but no Tb3+ luminescence was observed, indicating that Tb3+ binds to sites I and II but not sites III and IV. When two Tb3+ ions are mixed with 2Dy-calmodulin, Tb3+ luminescence rises rapidly as Tb3+ binds to the empty sites III and IV, followed by a more gradual decrease (k = 0.4 s-1 as the ions redistribute themselves over the four sites. These results indicate that (i) both Tb3+ and Dy3+ prefer binding to sites I and II of calmodulin and (ii) the binding of Tb3+ to calmodulin is not impeded by the presence of two Ca2+ ions initially bound to the protein. Thus, the Ca2+ and lanthanide ions must exhibit opposite preferences for the four sites of calmodulin: sites III and IV are the high-affinity sites for Ca2+, whereas Tb3+ and Dy3+ prefer sites I and II.     

Distance measurements in cardiac troponin C

Intramolecular distance measurements were made in cardiac troponin C (cTnC) by fluorescence energy transfer using Eu3+ or Tb3+ as energy donors and Nd3+ or an organic chromophore as acceptors. The laser-induced luminescence of bound Eu3+ is quenched in Eu1Nd1cTnC with a lifetime of 0.328 ms, compared with 0.43 ms for Eu2cTnC. The enhanced decay corresponds to an energy transfer efficiency of 0.25, or a distance of 1.1 nm between the two high affinity sites. We have also labeled cTnC with 4-dimethylaminophenylazophenyl-4'-maleimide (DAB-Mal) at the two cysteine residues (Cys-35 and Cys-84). Energy transfer measurements were carried out between Tb3+ bound to the high affinity sites and the labels attached to the domain containing the low affinity site. Upon uv irradiation at pH 6.7, Tb1cTnCDAB emits tyrosine-sensitized Tb3+ luminescence that decays bioexponentially with lifetimes of 1.29 and 0.76 ms. The shorter lifetime is ascribed to energy transfer from Tb3+ to the DAB labels, yielding an average distance of 3.4 nm between the donor and the acceptors. At pH 5.0, however, the luminescence decays exclusively with a single lifetime of 1.31 ms, suggesting that under these conditions all Tb3+ ions are more than 5.2 nm away from the label. Thus cTnC, like skeletal TnC, undergoes a pH-dependent conformational transition which converts an elongated structure at lower pH's to a rather compact conformation in a more physiological medium.     

The metal sites on sarcoplasmic reticulum membranes that bind lanthanide ions with the highest affinity are not the ATPase Ca2+ transport sites.

We attempted to establish whether lanthanide ions, when added to sarcoplasmic reticulum (SR) membranes in the absence of nucleotide, compete with Ca2+ for binding to the transport sites of the Ca(2+)-ATPase in these membranes, or whether they bind to different sites. Equilibrium measurements of the effect of lanthanide ions on the intrinsic fluorescence of SR ATPase and on 45Ca2+ binding to it were performed either at neutral pH (pH 6.8), i.e. when endogenous or contaminating Ca2+ was sufficient to nearly saturate the ATPase transport sites, or at acid pH (pH 5.5), which greatly reduced the affinity of calcium for its sites on the ATPase. These measurements did reveal apparent competition between Ca2+ and the lanthanide ions La3+, Gd3+, Pr3+, and Tb3+, which all behaved similarly, but this competition displayed unexpected features: lanthanide ions displaced Ca2+ with a moderate affinity and in a noncooperative way, and the pH dependence of this displacement was smaller than that of the Ca2+ binding to its own sites. Simultaneously, we directly measured the amount of Tb3+ bound to the ATPase relative to the amount of Ca2+ and found that Tb3+ ions only reduced significantly the amount of Ca2+ bound after a considerable number of Tb3+ ions had bound. Furthermore, when we tested the effect of Ca2+ on the amount of Tb3+ bound to the SR membranes, we found that the Tb3+ ions which bound at low Tb3+ concentrations were not displaced when Ca2+ was added at concentrations which saturated the Ca2+ transport sites. We conclude that the sites on SR ATPase to which lanthanide ions bind with the highest affinity are not the high affinity Ca2+ binding and transport sites. At higher concentrations, lanthanide ions did not appear to be able to replace Ca2+ ions and preserve the native structure of their binding pocket, as evaluated in rapid filtration measurements from the effect of moderate concentrations of lanthanide ions on the kinetics of Ca2+ dissociation. Thus, the presence of lanthanide ions slowed down the dissociation from its binding site of the first, superficially bound 45Ca2+ ion, instead of specifically preventing the dissociation of the deeply bound 45Ca2+ ion. These results highlight the need for caution when interpreting, in terms of calcium sites, experimental data collected using lanthanide ions as spectroscopic probes on SR membrane ATPase.     

Stoichiometry and location of terbium and calcium bindings to porcine intestinal calcium-binding protein

Quantitative analyses were carried out on Tb3+ binding to porcine intestinal calcium-binding protein (CaBP). Tb3+ (emission at 547 nm) and intrinsic tyrosine (emission at 303 nm) fluorescences upon excitation at 260 nm increase almost in parallel with increasing Tb3+ concentration up to a molar ratio of 2 against the protein in the CaBP solution. The pH dependence profile of Tb3+ fluorescence of the Tb3+-CaBP complex suggests that some free carboxylate groups are involved in the binding, as also suggested for Ca2+ binding. The results of fluorometric titration of Tb3+ and intrinsic tyrosine fluorescences of the CaBP complex with Tb3+ or Ca2+ led us to conclude that Tb3+ and Ca2+ have two common binding sites for each CaBP molecule. An equilibrium dialysis experiment showed that the dissociation constants of the two Tb3+-binding sites are 0.29 and 3.51 microM. Tb3+ strongly inhibits 45Ca binding to one of the two Ca2+-binding sites in the CaBP. All of these and previous results indicate that each Tb3+ ion can bind to either of two high-affinity Ca2+-binding sites in porcine intestinal CaBP with an affinity different from that for Ca2+ ion. We discuss the localization of the Ca2+- and Tb3+-binding sites in the CaBP.     

Co-operative binding of Ca2+ ions to the regulatory binding sites of gelsolin.

The rate of association of actin with gelsolin was measured at various Ca2+ and ATP concentrations. The fraction of Ca2+-activated gelsolin was determined by quantitative evaluation of the association rates thereby assuming that Ca2+-binding gelsolin associates with actin and Ca2+-free gelsolin does not. A plot of the fraction of Ca2+-activated gelsolin vs. the free Ca2+ concentration revealed a sigmoidal shape suggesting that co-operative binding of Ca2+ ions is required for activation of gelsolin. A good fit of the experimental data by calculated binding curves was obtained if two Ca2+ ions were assumed to bind to actin in a highly co-operative manner. ATP decreased the rate of association of gelsolin with actin and bound to gelsolin at a low affinity (Kd = 32 microm for Ca2+-free and Kd = 400 microm for Ca2+-activated gelsolin). In contrast, a 1 : 1 gelsolin-actin complex was found to be activated for association with actin by a single Ca2+ ion in a non-co-operative manner.     

Tb3+ binding to Ca2+ and Mg2+ binding sites on sarcoplasmic reticulum ATPase

The interactions of Tb3+ and sarcoplasmic reticulum (SR) were investigated by inhibition of Ca2+-activated ATPase activity and enhancement of Tb3+ fluorescence. Ca2+ protected against Tb3+ inhibition of SR ATPase activity. The apparent association constant for Ca2+, determined from the protection, was about 6 x 10(6) M-1, suggesting that Tb3+ inhibits the ATPase activity by binding to the high affinity Ca2+ binding sites. Mg2+ did not protect in the 2-20 mM range. The association constant for Tb3+ binding to this Ca2+ site was estimated to be about 1 x 10(9) M-1. No cooperativity was observed for Tb3+ binding. No enhancement of Tb3+ fluorescence was detected. A second group of binding sites, with weaker affinity for Tb3+, was observed by monitoring the enhancement of Tb3+ fluorescence (lambda ex 285 nm, lambda em 545 nm). The fluorescence intensity increased 950-fold due to binding. Ca2+ did not complete for binding at these sites, but Mg2+ did. The association constant for Mg2+ binding was 94 M-1, suggesting that this may be the site that catalyzes phosphorylation of the ATPase by inorganic phosphate. For vesicles, Tb3+ binding to these Mg2+ sites was best described as binding to two classes of binding sites with negative cooperativity. If the SR ATPase was solubilized in the nonionic detergent C12E9 (dodecyl nonaoxyethylene ether alcohol), in the absence of Ca2+, only one class of Tb3+ binding sites was observed. The total number of sites appeared to remain constant. If Ca2+ was included in the solubilization step, Tb3+ binding to these Mg2+ binding sites displayed positive cooperativity (Hill coefficient, 2.1). In all cases, the apparent association constant for Tb3+, in the presence of 5 mM MgCl2, was in the range of 1-5 x 10(4) M-1.     

Europium(III) luminescence and tyrosine to terbium(III) energy-transfer studies of invertebrate (octopus) calmodulin.

The effects of minor differences in the amino acid sequences between a vertebrate (bovine testes) and an invertebrate (octopus) calmodulin on metal ion binding were investigated via laser-induced Eu3+ and Tb3+ luminescence. Amino acid substitutions at residues which are coordinated to the metal ion do not produce any detectable changes in the 7F0----5D0 excitation spectrum of the Eu3+ ion bound to octopus calmodulin relative to bovine testes calmodulin; only minor differences in the excited-state lifetime values in D2O solution are observed. The dissociation constants for Eu3+ (1.0 +/- 0.2 microM) and Tb3+ (5 +/- 1 microM) from the weak lanthanide binding sites (III and IV, numbered from the amino terminus) of octopus calmodulin were measured using luminescence techniques. Both values agree well with those reported previously for bovine testes calmodulin [Mulqueen, P. M., Tingey, J. M., & Horrocks, W. D., Jr. (1985) Biochemistry 24, 6639-6645]. The measured dissociation constant of Eu3+ bound in the tight lanthanide binding sites (I and II) is 6 +/- 2 nM for octopus calmodulin and 12 +/- 2 nM for bovine testes calmodulin. The distances between sites I and II (12.4 +/- 0.5 A) and sites III and IV (11.7 +/- 0.8 A) were determined from F?rster-type energy transfer in D2O solutions of octopus calmodulin containing bound Eu3+ donor and Nd3+ acceptor ions. F?rster theory parameters for nonradiative energy transfer between Tyr138 and Tb3+ ions bound at sites III and IV of octopus calmodulin were comprehensively evaluated, including a dynamics simulation of the orientation factor kappa 2. This theory is found to account quantitatively for the observed energy-transfer efficiency as evaluated from the observed sensitized Tb3+ emission.     

Terbium(III) luminescence study of the spatial relationship of tryptophan residues to the two metal ion binding sites of Escherichia coli glutamine synthetase.

The luminescence of Tb(III) was used to explore the topography of the metal ion sites of Escherichia coli glutamine synthetase and the relationship between these sites and tryptophan residues of the enzyme. By irradiation of tryptophan residues at 295 nm and measurement of the resulting Tb(III) luminescence at 544 nm, a biphasic curve was obtained upon titrating apoenzyme with Tb(III) indicating sequential binding of Tb(III) ions to the two binding sites of glutamine synthetase. The luminescence intensity was greater in the second region of the titration curve which is mostly due to energy transfer from Trp-158 to the second Tb(III) binding site of the enzyme. By use of the F?rster equation for energy transfer from donor Trp to acceptor Tb(III), distances from Trp-57 to Tb(III) at the n1 and n2 sites were calculated, by using a mutant enzyme in which Trp-158 was replaced by Ser, to be 16.4 and 15.7 A, respectively; distances from Trp-158 to Tb(III) at the n1 and n2 sites were calculated, by using a mutant enzyme in which Trp-57 was replaced by Leu, to be 16.8 and 9.5 A, respectively. All the distances are in reasonably good agreement with the crystal structure distances from Salmonella typhimurium glutamine synthetase except the distance from Trp-158 to the second Tb(III) binding site. The discrepancies may result from a slightly different conformation of glutamine synthetase in solution and in the crystal and/or a slightly different conformation for trivalent Ln(III) binding compared to divalent Mn(II) binding.     

Luminescence of peptide-bound terbium ions. Determination of binding constants

Luminescence of Tb3+ ions bound to a calmodulin fragment has been studied. It is shown that during their lifetime excited ions dissociate from the peptide. If concentration of free peptide is high enough they can be coordinated again. As a consequence, observed terbium luminescence lifetime and intensity depends not only on binding equilibrium, but also on concentration of free peptide molecules. In such a system terbium binding constant cannot be correctly determined by simple steady-state measurements of luminescence intensities. Instead, terbium luminescence decay curves measured at various peptide concentrations must be analysed. Such an analysis has been made for a fragment of the IIIrd calcium binding domain of rat testis calmodulin. Rate constant of terbium association and the equilibrium binding constant corresponding to the best fit of theoretical functions to experimental points have been determined.     

Luminescence studies of lanthanide ion binding to parvalbumin

Luminescence methods were used to examine the interaction of Eu(III) and Tb(III) with parvalbumin isozyme III from pike (Esox lucius). The bound lanthanide ions were excited both directly, via laser irradiation, and indirectly, via fluorescence energy transfer from adjacent phenylalanine residues. At high (175 microM) protein concentrations, the lanthanide titration curves exhibited pronounced quenching of luminescence at Ln3+:parvalbumin ratios above 2:1, in agreement with earlier reports (Donato, H., Jr., and Martin, R. B. (1974) Biochemistry 13, 4575-4579). However, in experiments performed with lower concentrations (10 microM), the titrations were well behaved and indicated a lanthanide:protein stoichiometry of 2:1. Equilibrium dialysis measurements performed with Eu(III) ruled out the existence of a third strong binding site which could cause the quenching of the luminescence at high protein concentrations. Similarly, careful analysis of the spectrum that results from direct excitation of the 7F0----5D0 transition of parvalbumin-bound Eu3+ ion revealed no peak attributable to a third Ln3+-binding site. The peak which has been construed by others (Rhee, M.-J., Sudnick, D. R., Arkle, V. K., and Horrocks, W. DeW., Jr. (1981) Biochemistry 20, 3328-3334) as evidence for a third site was shown to result from a pH-dependent spectral transition involving the europium ions bound at the CD and EF sites. Luminescent lifetime measurements performed on Tb(III)/parvalbumin solutions follow Stern-Volmer quenching kinetics at terbium:protein ratios in excess of 2:1, suggesting that the quenching results from collisional deactivation of the tightly bound ions by excess terbium ion free in solution.     

Spectroscopic studies of metal ion binding to a tryptophan-containing parvalbumin

The binding of Ca(II) and members of the trivalent lanthanide ion, Ln(III), series to apoparvalbumin (isotype pI = 4.75) from codfish (Gadus callarius L) results in the development of a distinctive sharp feature in the UV absorption spectrum at about 290 nm. Titration curves obtained by monitoring the spectral change in this region reveal a change in slope after the addition of 1 equiv of metal ion and no further rise after 2 equiv has been added, consistent with sequential binding to the principal EF and CD sites. Laser-induced luminescence excitation spectra of the 7F0----5D0 transition of bound Eu(III) demonstrate the quantitative binding of this ion to the principal sites and disclose the presence of a subsidiary site at pH values greater than 6. Metal ion competition experiments monitored by means of this excitation transition show that the early members of the Ln(III) ion series bind more tightly than those at the end. Tryptophan-sensitized Tb(III) luminescence reveals that this ion binds sequentially to the EF and CD sites, in that order. The intrinsic tryptophan fluorescence of apoparvalbumin is increased in a stepwise fashion as Ca(II) or Ln(III) ions bind sequentially, with the exceptions of Eu(III) and Yb(III). The binding of the latter two ions causes quenching of the protein fluorescence via an energy-transfer process which involves low-lying charge-transfer bands. The distance dependences of the tryptophan to Tb(III) and tryptophan to Eu(III) energy-transfer processes are observed to be identical, consistent with a F?rster-type mechanism in both cases.     

Characterization of Gd3+ and Tb3+ binding sites on Ca2+,Mg2+-adenosine triphosphatase of sarcoplasmic reticulum

Interaction between Gd3+ and Tb3+ ions and Ca2+,Mg2+-ATPase of sarcoplasmic reticulum was studied. Three classes of lanthanide-ion binding sites with different affinities were distinguished. Binding of Gd3+ to the site with the highest affinity seemed to occur at less than 10(-6)M free Gd3+ and resulted in severe inhibition of ATPase activity. The reaction rates of both E-P formation and decomposition in the forward direction were inhibited in parallel with this binding, whereas ADP-dependent decay of E-P in the backward direction was not. At these Gd3+ concentrations, Ca2+-binding to the transport site was not inhibited. Binding of Gd3+ and Tb3+ to the Ca2+-transport site did occur, but more than 10(-5)M free Gd3+ or Tb3+ was required for effective competition with Ca2+ for that site. Gd3+ bound to the transport site in place of Ca2+ did not activate the E-P intermediate formation. Addition of 10(-1)M Tb3+ to a suspension of sarcoplasmic reticulum membranes resulted in marked enhancement of Tb3+ fluorescence, which is due to an energy transfer from aromatic amino acid residues of ATPase to Tb3+ ions bound to the low affinity site of the enzyme. Gd3+ and Mn2+ competed with Tb3+ for that site, but Ca2+, Zn2+, and Cd2+ did not.     

Ca2+ regulation of gelsolin activity: binding and severing of F-actin.

Regulation of the F-actin severing activity of gelsolin by Ca2+ has been investigated under physiologic ionic conditions. Tryptophan fluorescence intensity measurements indicate that gelsolin contains at least two Ca2+ binding sites with affinities of 2.5 x 10(7) M-1 and 1.5 x 10(5) M-1. At F-actin and gelsolin concentrations in the range of those found intracellularly, gelsolin is able to bind F-actin with half-maximum binding at 0.14 microM free Ca2+ concentration. Steady-state measurements of gelsolin-induced actin depolymerization suggest that half-maximum depolymerization occurs at approximately 0.4 microM free Ca2+ concentration. Dynamic light scattering measurements of the translational diffusion coefficient for actin filaments and nucleated polymerization assays for number concentration of actin filaments both indicate that severing of F-actin occurs slowly at micromolar free Ca2+ concentrations. The data suggest that binding of Ca2+ to the gelsolin-F-actin complex is the rate-limiting step for F-actin severing by gelsolin; this Ca2+ binding event is a committed step that results in a Ca2+ ion bound at a high-affinity, EGTA-resistant site. The very high affinity of gelsolin for the barbed end of an actin filament drives the binding reaction equilibrium toward completion under conditions where the reaction rate is slow.     

Comparison of terbium (III) luminescence enhancement in mutants of EF hand calcium binding proteins.

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 F?rster equation in approximating distances in these systems.     

Distance measurements between metal-binding sites of calmodulin and from these sites to Cys-133 of troponin I in the binary complex

Distances between the four Ca2+-binding sites of calmodulin (CaM) have been measured by fluorescence energy transfer techniques using Eu3+ and Tb3+ as energy donors and a number of other lanthanide ions (Ln3+) as acceptors. It was shown previously that lanthanide ions preferentially bind to sites I and II of CaM with an affinity higher than that for sites III and IV (Kilhoffer, M.-C., Demaille, J. G., and Gerald, D. (1980) FEBS Lett. 116, 269-272; Wang, C.-L. A., Aquaron, R. R., Leavis, P. C., and Gergely, J. (1982) Eur. J. Biochem. 124, 7-12). Thus upon direct excitation with a laser the luminescence lifetimes of Eu1Ln1CaM and Tb1Ln1CaM provide information on the distance between sites I and II. On the other hand, since Tb3+ ions bound to sites III and IV are sensitizable through tyrosine residues, lifetime measurements of Tb2Ln2CaM excited by UV light yield the distance between sites III and IV. Both pairs of sites were found to be separated by a distance of 1.05 +/- 0.07 nm. Binding of Ca2+ to sites III and IV does not alter the distance between sites I and II. We have also attached a chromophoric label, dimethylaminophenylazobenzene, to Cys-133 of skeletal troponin I and carried out distance measurements on its complex with CaM by both direct and indirect excitation. The averaged distances from sites I and II in the N-terminal half and from sites III and IV in the C-terminal half of the CaM molecule to the label on troponin I are 2.7 and 2.5 nm, respectively.     

Metal ion binding sites of bacteriorhodopsin. Laser-induced lanthanide luminescence study

Laser-excited luminescence lifetimes of lanthanide ions bound to bacteriorhodopsin have been measured in deionized membranes. The luminescence titration curve, as well as the binding curve of apomembrane (retinal-free) with Eu3+, has shown that the removal of the retinal does not significantly affect the affinity of Eu3+ for the two high affinity sites of bacteriorhodopsin. The D2O effects on decay rate constants indicate that Eu3+ bound to the high affinity sites of native membrane or apomembrane is coordinated by about six ligands in the first coordination sphere. Tb3+ is shown to be coordinated by four ligands. The data indicate that metal ions bind to the protein with a specific geometry. From intermetal energy transfer experiments using Eu3+-Pr3+, Tb3+-Ho3+, and Tb3+-Er3+, the distance between the two high affinity sites is estimated to be 7-8 A.     

Interactions of adriamycin with a calcium binding site

Terbium (Tb3+) luminescence has been used to investigate the interactions of adriamycin with a specific calcium binding protein, in the plasma membrane of GH3/B6 pituitary tumor cells. The luminescence intensity and lifetime of the Tb3+-GH3/B6 complex was quenched in the presence of adriamycin. According to Stern-Volmer analysis, the quenching of Tb3+-GH3/B6 luminescence was by both membrane bound adriamycin (Ka = 3.7 x 10(5) M-1) and free adriamycin (kq = 7.3 x 10(7) M-1 s-1). The data suggests that, the calcium binding site at the outer surface of the membrane is collisionally accessible to freely diffusing adriamycin; and, that the toxin receptor site is located near the bound metal ion.     

Purification and properties of a novel nucleotide-hydrolysing enzyme (5''-nucleotidase) from Boophilus microplus.

Conformational changes in the beta-subunit of the bovine brain Ca2+-binding protein S100b (S100-beta) accompanying Ca2+ binding were investigated by analysis of the spectroscopic properties of the single tyrosine residue (Tyr17 beta) and flow-dialysis binding experiments. S100-beta binds Ca2+ sequentially at two sites to change the conformation of the protein. The first Ca2+ ion binds to site II beta, a typical Ca2+-binding site in the C-terminal region, and it does not significantly perturb the proximal environment of Tyr17 beta. After the first site is occupied, another Ca2+ ion binds to the N-terminal Ca2+-binding site, I beta, and strengthens a hydrogen bond between Tyr17 beta and a neighbouring carboxylate acceptor group, which results in a large increase in the Tyr17 beta fluorescence spectrum half-width and a positive absorption and c.d. signal between 290 and 275 nm. Ca2+ binding to the S100b.Zn2+6 complex, studied by flow-dialysis and fluorescence measurements showed that, although Zn2+ ions increase the affinity of S100b protein for Ca2+, the Ca2+-binding sequence was not changed. Tb3+ (terbium ion) binding studies on the S100b.Zn2+6 complex proved that Tb3+ antagonizes only Ca2+ binding site II beta and confirmed the sequential occupation of Ca2+-binding sites on the S100b.Zn2+6 complex.