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.     

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 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.     

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 interaction of cyclosporin and calmodulin

The interaction of cyclosporin A and dansyl cyclosporin A with bovine and wheat germ calmodulin has been monitored by measurements of induced changes in dansyl and bound toluidinyl naphthalene sulfonate fluorescence. The interaction is Ca2(+)-dependent and 1:1. Measurements of the efficiency of radiationless energy transfer from bound dansyl cyclosporin A to an acceptor group located on Cys-27 of wheat germ calmodulin suggest that the primary binding site is not located on the N-terminal lobe (residues 1-65). However, studies with proteolytic fragments of calmodulin indicate that elements of the N-terminal half-molecule (residues 1-77) may be involved in the stabilization of the binding site. The binding of cyclosporin alters the physical properties of calmodulin and, in particular, reduces the localized rotational mobility of a fluorescent probe.     

Lanthanide ions and Cd2+ are able to substitute for Ca2+ in regulating phosphorylase kinase

Trivalent lanthanide ions and Cd2+ were found to mimic effectively the stimulatory action of Ca2+ on rabbit muscle phosphorylase kinase. In the range of concentrations tested, Cd2+ and lanthanides (Tb3+, Gd3+, Pr3+, Ce3+) could substitute for Ca2+ in activating the enzyme to about 60% and 70% respectively of the maximal level seen with Ca2+, at pH 8.2. The effect induced by Cd2+ was biphasic (stimulation followed by inhibition with increasing metal cation concentration). Similar results were obtained at pH 6.8. Cd2+ and Tb3+ were also able to replace Ca2+ required for the stimulation of phosphorylase kinase activity at pH 8.2 by exogenous calmodulin. Maximal stimulation induced by calmodulin in presence of Cd2+ was significantly higher than that in presence of Ca2+ or Tb3+.     

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.     

Effects of terbium and pH on structure of anticoagulation factor II from Agkistrodon acutus venom probed by fluorescent spectroscopy and equilibrium dialysis

Anticoagulation factor II (ACF II) isolated from the venom of Agkistrodon acutus is an activated coagulation factor X-binding protein with marked anticoagulant activity. Present studies show that the pH has a marked effect on the fluorescence intensity of holo-ACF II; however, no appreciable shift of the emission maximum of holo-ACF II was observed in the pH range of 3-10. It was deduced from a relatively weak fluorescence emission of holo-ACF II at a neutral pH (6-7) that native holo-ACF II assumes a compactly folded structure in which the most interior Trp residues and quenchers are adjacent. Terbium ions can completely replace both Ca2+ ions in holo-ACF II as determined by equilibrium dialysis. Two Tb3+-binding sites with different apparent Tb3+ association constant values, (2.1 +/- 0.2) and (1.0 +/- 0.1) x 10(7) M(-1), were identified through Tb3+ fluorescence titration. In addition, it was confirmed from the titration of holo-ACF II and Tb3+-ACF II with N-bromosuccinimide (NBS) that only interior Trp residues are involved in the energy transfer to Tb3+ ions and all accessible Trp residues located in the surface of holo-ACF II have a similar affinity to NBS while those located in the surface of Tb3+-ACF II have two different kinds of affinity to NBS, which suggests a conformational change of holo-ACF II on the substitution of Tb3+ for Ca2+.     

NMR approaches for monitoring domain orientations in calcium-binding proteins in solution using partial replacement of Ca2+ by Tb3+.

This work shows that the partial replacement of diamagnetic Ca2+ by paramagnetic Tb3+ in Ca2+/calmodulin systems in solution allows the measurement of interdomain NMR pseudocontact shifts and leads to magnetic alignment of the molecule such that significant residual dipolar couplings can be measured. Both these parameters can be used to provide structural information. Species in which Tb3+ ions are bound to only one domain of calmodulin (the N-domain) and Ca2+ ions to the other (the C-domain) provide convenient systems for measuring these parameters. The nuclei in the C-domain experience the local magnetic field induced by the paramagnetic Tb3+ ions bound to the other domain at distances of over 40 A from the Tb3+ ion, shifting the resonances for these nuclei. In addition, the Tb3+ ions bound to the N-domain of calmodulin greatly enhance the magnetic susceptibility anisotropy of the molecule so that a certain degree of alignment is produced due to interaction with the external magnetic field. In this way, dipolar couplings between nuclear spins are not averaged to zero due to solution molecular tumbling and yield dipolar coupling contributions to, for example, the one-bond 15N-1H splittings of up to 17 Hz in magnitude. The degree of alignment of the C-domain will also depend on the degree of orientational freedom of this domain with respect to the N-domain containing the Tb3+ ions. Pseudocontact shifts for NH groups and 1H-15N residual dipolar couplings for the directly bonded atoms have been measured for calmodulin itself, where the domains have orientational freedom, and for the complex of calmodulin with a target peptide from skeletal muscle myosin light chain kinase, where the domains have fixed orientations with respect to each other. The simultaneous measurements of these parameters for systems with domains in fixed orientations show great potential for the determination of the relative orientation of the domains.     

Tb~(3+)作为荧光探针研究钙调蛋白与拮抗药物的相互作用

本文报导以Tb~(3+)作为荧光探针,研究钙调蛋白(CaM)与其拮抗药物分子间相互作用的机制.所用方法简便、快速、灵敏.CaM的内源荧光研究表明,Tb~(3+)类似于Ca~(2+),也能诱导CaM分子构象发生改化,由于CaM分子中Ca~(2+)的第Ⅲ、Ⅳ结合位点上各有一个Tyr线基,如(?)280nm激发,则发生从Tyr向Tb~(3+)的能量转移,从而导致Tb~(3+)在490和545nm处的特征荧光发射大大加强.本文检测了药物分子与Tb~(3+)-CaM结合对该荧光发射的影响.实验表明,TFP与CaM的高亲和位点处于CaM分子C-末端部位,即含第Ⅲ、Ⅳ结构域的半分子上:丙拮抗药物酸枣仁皂甙A则优先结合在含第Ⅰ、Ⅱ的结构域的另一半分子(?).     

Calcium binding domains of calmodulin. Sequence of fill as determined with terbium luminescence

Terbium, a trivalent lanthanide, effectively substituted for Ca2+ in calmodulin as judged by several criteria: intrinsic fluorescence spectra, altered mobilities on polyacrylamide gel electrophoresis, formation of a stable complex with troponin I or calcineurin, and stimulation of phosphodiesterase. Calmodulin harbors four Ca2+ binding domains; domains I and II contain no tyrosine, whereas domains III and IV each have one tyrosine. The binding of Tb3+ to calmodulin was followed by the increase of Tb3+ fluorescence at 545 nm upon binding to calmodulin. This fluorescence was elicited either by exciting Tb3+ directly at 222 nm or by exciting the calmodulin tyrosine at 280 nm with resulting energy transfer from tyrosine to Tb3+. Fluorescence generated by direct excitation measures binding of Tb3+ to any of the Ca2+ binding domains, whereas energy transfer through indirect excitation is effective only when Tb3+ is within 5 A of tyrosine, indicating that Tb3+ necessarily occupies a Ca2+ binding domain that contains tyrosine. A judicious use of the direct and indirect excitation could reveal the sequence of fill of the binding domains. Our results suggest these domains are filled in the following sequence: 1) domain I or II; 2) domains III and IV; and 3) domain II or I that has not been filled initially.     

The intrinsic tryptophan fluorescence of beta 1-bungarotoxin and the Ca2+-binding domains of the toxin as probed with Tb3+ luminescence.

beta 1-Bungarotoxin has only one tryptophan residue, namely Trp-19 in the phospholipase A2 subunit. The environment of Trp-19 was studied by intrinsic fluorescence and solute quenching. The native protein showed an emission peak at 330 nm. About 90% of the fluorescent tryptophan was accessible to quenching by either acrylamide or KI but not to CsCl. A red-shift in the emission peak occurred between 2.0 M- and 4.0 M-guanidinium chloride, and the helix-coil transition of the polypeptide backbone occurred between 4.0 M- and 6.0 M-guanidinium chloride. These results suggested that Trp-19 was in a less polar medium but near a positive charge. The local conformation around Trp-19 could be disturbed by binding of Tb3+ or Ca2+ or Sr2+ to the toxin molecule. Tb3+ a tervalent lanthanide ion, effectively substituted for Ca2+ in stimulating the phospholipase A2 activity of beta 1-bungarotoxin. Upon the binding of Tb3+ to the toxin, the Tb3+ fluorescence in the 450-650 nm region was enhanced. This resulted from the energy transfer from Trp-19 to Tb3+. The distance between the energy-transfer pair was estimated to be 0.376-0.473 nm at pH 7.6 and 0.486-0.609 nm at pH 6.3. Assuming that there were two Tb3+-binding sites on the toxin molecule, at pH 7.6 the association constants of the high-affinity and the low-affinity sites were determined to be 3.82 x 10(3) M-1 and 2.85 x 10(2) M-1 respectively. At between pH 6.0 and 7.0 Tb3+ bound to the high-affinity site decreased greatly but did not disappear entirely. Both Ca2+ and Sr2+ competed with Tb3+ at the high-affinity sites, but Sr2+ could not substitute for Ca2+ in stimulating the phospholipase A2 activity.     

Analysis of aluminum and divalent cation binding to wheat root plasma membrane proteins using terbium phosphorescence

A phosphorescent trivalent cation, terbium [Tb(III)], has been used to study the binding of different polyvalent cations to the proteins of wheat (Triticum aestivum L.) root plasma membranes. The phosphorescence emission intensity of Tb(III) was enhanced after Tb(III) binding to wheat root plasma membranes as a result of nonradiative resonance energy transfer from the membrane protein tyrosine and phenylalanine residues. Complex, saturable Tb(III) binding was observed, suggesting multiple binding sites. Bound Tb(III) could be displaced by divalent cations in the general order: Mn(II) > Ca(II) > Mg(II). Al(III) was very effective in reducing the protein-enhanced Tb(III) phosphorescence at pH values below 5. Al(III) also altered the Tb(III) phosphorescence lifetime, suggesting Al(III)-induced changes in membrane protein conformation. The more Al(III)-sensitive wheat cultivar (Anza) bound Al(III) with higher affinity than the more tolerant cultivar (BH 1146). At pH 5.5 where Al(III) did not displace bound Tb(III), low levels of Al(III) reduced the ability of Mn(II) to decrease Tb(III) phosphorescence. The significance of these results is discussed with respect to the mechanisms of Al(III) tolerance in wheat and the potential beneficial effects of Al(III) in reducing Mn(II) phytotoxicity.     

Localization of a fibrinogen calcium binding site between gamma-subunit positions 311 and 336 by terbium fluorescence

Calcium is required for effective fibrin polymerization. The high affinity Ca2+ binding capacity of fibrinogen was directly localized to the gamma-chain by autoradiography of nitrocellulose membrane blots of fibrinogen subunits incubated with 45Ca2+. Terbium (Tb3+) competitively inhibited 45Ca2+ binding to fibrinogen during equilibrium dialysis, accelerated fibrin polymerization, and limited fibrinogen fragment D digestion by plasmin. The intrinsic fluorescence of Ca2+-depleted fibrinogen was maximally enhanced by Ca2+ and Tb3+, but not by Mg2+, at about 3 mol of cation/mol of fibrinogen. Protein-bound Tb3+ fluorescence at 545 nm was maximally enhanced by resonance energy transfer from tryptophan (excitation at 290 nm) at about 2 mol of Tb3+mol of fibrinogen and about 1 mol of Tb3+/mol of plasmic fragment D94 (Mr 94,000). Fibrinogen fragments D78 (Mr 78,000) and E did not show effective enhancement of Tb3+ fluorescence, suggesting that the Ca2+ site is located within gamma 303 to gamma 411, the peptide which is absent in fragment D78 but present in D94. When CNBr fragments of the carboxyamidated gamma-subunit were assayed for enhancement of Tb3+ fluorescence, peptide CBi (gamma 311-336) bound 1 mol of Tb3+/mol of CBi. Thus, the Ca2+ site is located within this peptide. The sequence between gamma 315 and gamma 329 is homologous to the calmodulin and parvalbumin Ca2+ binding sites.     

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.     

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.     

Distances between the functional sites of the (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum

Luminescence energy transfer measurements have been used to determine the distances between the two high affinity Ca2+ binding-transport sites of the (Ca2+ + Mg2+)-ATPase of skeletal muscle sarcoplasmic reticulum. The lanthanide Tb3+ situated at one high affinity Ca2+ site was used as the transfer donor, and acceptors at the other Ca2+ site were the lanthanides Nd3+, Pr3+, Ho3+, or Er3+. Terbium bound to the enzyme was excited directly with a pulsed dye laser. Analysis of the changes in the terbium luminescence lifetime due to the presence of the acceptor indicates that the distance between the Ca2+ sites is 10.7 A. The distance between the Ca2+ sites and the nucleotide-binding catalytic site was determined using Tb3+ at the Ca2+ sites and either trinitrophenyl nucleotides (TNP-N) or fluorescein 5-isothiocyanate (FITC) in the catalytic site as energy acceptors. The R0 values for the Tb-acceptor pairs are approximately 30 and approximately 40 A for TNP-N and FITC, respectively. The distance between Tb3+ at the Ca2+ sites and TNP-ATP at the nucleotide site is approximately 35 A and that between the Ca2+ sites and the FITC labeling site is approximately 47 A. Considerations of the molecular dimensions of the ATPase polypeptide indicate that while the two Ca2+ sites are close to each other, the Ca2+ sites and the nucleotide site are quite remote in the three-dimensional structure of the enzyme.     

Nicotinic acetylcholine receptor channel electrostatics determined by diffusion-enhanced luminescence energy transfer

The electrostatic potentials within the pore of the nicotinic acetylcholine receptor (nAChR) were determined using lanthanide-based diffusion-enhanced fluorescence energy transfer experiments. Freely diffusing Tb3+ -chelates of varying charge constituted a set of energy transfer donors to the acceptor, crystal violet, a noncompetitive antagonist of the nAChR. Energy transfer from a neutral Tb3+ -chelate to nAChR-bound crystal violet was reduced 95% relative to the energy transfer to free crystal violet. This result indicated that crystal violet was strongly shielded from solvent when bound to the nAChR. Comparison of energy transfer from positively and negatively charged chelates indicate negative electrostatic potentials of -25 mV in the channel, measured in low ionic strength, and -10 mV measured in physiological ionic strength. Debye-Hückel analyses of potentials determined at various ionic strengths were consistent with 1-2 negative charges within 8 A of the crystal violet binding site. To complement the energy transfer experiments, the influence of pH and ionic strength on the binding of [3H]phencyclidine were determined. The ionic strength dependence of binding affinity was consistent with -3.3 charges within 8 A of the binding site, according to Debye-Hückel analysis. The pH dependence of binding had an apparent pKa of 7.2, a value indicative of a potential near -170 mV if the titratable residues are constituted of aspartates and glutamates. It is concluded that long-range potentials are small and likely contribute little to selectivity or conductance whereas close interactions are more likely to contribute to electrostatic stabilization of ions and binding of noncompetitive antagonists within the channel.     

The binding of terbium ions to gelsolin reveals two classes of metal ion binding sites.

Spectroscopically active terbium ions have been used to probe the Ca2+ ion-binding sites on human plasma gelsolin. The luminescence of Tb3+ ions bound to gelsolin is markedly enhanced when excited indirectly at 295 nm due to F?rster type dipole-dipole energy transfer from neighboring tryptophan residues. Titration of this luminescence with increasing concentrations of Tb3+ ions was saturable although the shape of this titration curve was complex indicating the involvement of multiple classes of sites. Luminescence lifetime measurements (obtained by indirect excitation at 295 nm) demonstrate the presence of two classes of sites characterized by a major lifetime of 1.0-1.1 ms and a minor lifetime of 0.7-0.8 ms. However, while the amplitude of the minor lifetime showed a hyperbolic dependence on the Tb3+ ion concentration, the amplitude of the major lifetime showed a strongly sigmoidal dependence. Different classes of Tb3+ ion binding sites can also be distinguished by the different Ca2+ ion concentrations needed to displace Tb3+ ions from these sites on gelsolin. It is proposed that the occupancy of one class of Tb3+ ion binding sites on gelsolin causes a conformational change in gelsolin which then allows a second class of cryptic Tb3+ ion binding sites to be expressed. The implications of these results in terms of the binding of Ca2+ ions to gelsolin and the regulation of the activities of gelsolin by calcium are discussed.     

Ca2(+)-induced conformational change and aggregation of chromogranin A

Chromogranin A, the most abundant protein in bovine adrenal chromaffin granules, bound calmodulin in a Ca2(+)-dependent manner, and the calmodulin-binding property was utilized to purify chromogranin A. Chromogranin A has been described in the past as a "random-coil polypeptide" with little alpha-helix or beta-sheet conformation. However, circular dichroism measurements with pure, native chromogranin A revealed relatively high alpha-helical contents (40% at the intravesicular pH of 5.5). Fluorescence studies confirmed previous observations that chromogranin A binds Ca2+ with low affinity. Considering the high concentration of Ca2+ in the secretory vesicle, the effect of Ca2+ on the secondary structure and self-association of chromogranin A was examined. Ca2+ induced a decrease of alpha-helicity of chromogranin A from 40 to 30% at pH 5.5. In contrast, at pH 7.5 the same amount of Ca2+ increased alpha-helicity of the protein from 25 to 40%. Boiling of the adrenal extract, a commonly used purification procedure for chromogranin A, resulted in the isolation of conformationally distinct chromogranin A molecule. Unlike secretory protein-I of the parathyroid gland (Gorr, S.-V., Dean, W. L., Radley, T. L., and Cohn, D. V. (1988) Bone Mineral 4, 17-25), chromogranin A aggregated rapidly in the presence of Ca2+. The extent and rate of aggregation were highly dependent on Ca2+ concentration. Although both the rate and extent of aggregation at pH 7.5 were much lower than those at pH 5.5, aggregation of chromogranin A proceeded at both pH's. In this respect, chromogranin A differs from human chromogranin C which was shown by Gerdes et al. (Gerdes, H.-H., Rosa, P., Phillips, E., Baeuerle, P. A., Frank, R., Argos, P., and Huttner, W. B. (1989) J. Biol. Chem. 264, 12009-12015) to aggregate at pH 5.2 but not at pH 7.4.