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.     

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.     

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.     

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+.     

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.     

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(?).     

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.     

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.     

Interaction of lanthanide ions with bovine factor X and their use in the affinity chromatography of the venom coagulant protein of Vipera russelli.

The substitution of trivalent lanthanide ions for Ca(II) in the Ca(II)-DEPENDENT ACTIVATION OF BOVINE Factor X by the coagulant protein of Russell's viper venom was studied at pH 6.8. Factor X contains two high affinity metal binding sites which bind Gd(III), Sm(III), and Yb(III) with a Kd of about 4 X 10-7 M and four to six lower affinity metal binding sites which bind Gd(III), Sm(III) with a Kd of about 1.5 X 10-5M. In comparison, 1 mol of Factor X binds 2 mol of Ca(II) with a Kd of 3 X 10-4M and weakly binds many additional Ca(II) ions. No binding of Gd(III) to the venom protein was observed. Dy(III), Yb(III), Tb(III), Gd(III), Eu(III), La(III), AND Nd(III) cannot substitute for Ca(II) in the Ca(II)-dependent activation of Factor X by the venom protein at pH 6.8. Kinetic data consistent with the models of competitive inhibition of Ca(II) by Nd(III) yielded a Ki of 1 to 4 X 10-6M. The substitution of lanthanide ions for Ca(II) to promote protein complex formation of Factor X-metal-venom protein without the activation of Factor X facilitated the purification of the coagulant protein from crude venom by affinity chromatography. Using a column containing Factor X covalently bound to agarose which was equilibrated in 10 mM Nd(III), Tb(III), Gd(III), or La(III), the coagulant protein was purified 10-fold in 40% yield from crude venom and migrated as a single band on gel electrophoresis in sodium dodecyl sulfate. These data suggest that lanthanide ions complete with Ca(II) for the metal binding sites of Factor X and facilitate the formation of a nonproductive ternary complex of venom protein-Factor X-metal. Tb(III) fluorescence, with emission maxima at 490 and 545 nm, is enhanced 10,000-fold in the presence of Factor X. The study of the participation of an energy donor intrinsic to Factor X in energy transfer to Tb(III) may be useful in the characterization of the metal binding sites of Factor X.     

Characterization of lanthanide (III) ion binding to calmodulin using luminescence spectroscopy

Pulsed dye laser excitation spectroscopy of the 7F0----5D0 transition of Eu(III) reveals only a single peak as this ion is titrated into apocalmodulin. A titration based on the intensity of this transition shows that the first two Eu(III) ions bind quantitatively to two tight sites, followed by weaker binding (Kd = 2 microM) to two additional sites under conditions of high ionic strength (0.5 M KC1). This excitation experiment is also shown to be a general method for measuring contaminating levels of EDTA down to 0.2 microM in proton solutions. Experiments with Tb(III) using both direct laser excitation and indirect sensitization of Tb(III) luminescence through tyrosine residues in calmodulin also give evidence for two tight and two weaker binding sites (Kd = 2-3 microM). The indirect sensitization results primarily upon binding to the two weaker sites, implying that Tb(III) binds first to domains I and II, which are remote from tyrosine-containing domains III and IV. The 7F0----5D0 excitation signal of Eu(III) was used to measure the relative overall affinities of the tripositive lanthanide ions, Ln(III), across the series. Ln(III) ions at the end of the series are found to bind more weakly than those at the beginning and middle of the series. Eu(III) excited-state lifetime measurements in H2O and D2O reveal that two water molecules are coordinated to the Eu(III) at each of the four metal ion binding sites. Measurements of F?rster-type nonradiative energy-transfer efficiencies between Eu(III) and Nd(III) in the two tight sites were carried out by monitoring the excited-state lifetimes of Eu(III) in the presence and absence of the energy acceptor ion Nd(III).(ABSTRACT TRUNCATED AT 250 WORDS)     

Time-resolved europium(III) luminescence excitation spectroscopy: characterization of calcium-binding sites of calmodulin

Pulsed-dye laser excitation and lifetime spectroscopy of the 7F0----5D0 transition of Eu3+ reveals details of the binding of this ion to the calcium-binding sites of calmodulin (labeled I-IV, starting at the N-terminus). For 10 microM calmodulin Eu3+ binds quantitatively at sites I and II and more weakly at sites III and IV with Kd values of approximately 0.5 microM and 1.0 microM at the latter sites. In D2O solution the time course of luminescence emission of Eu3+-loaded calmodulin can be separated into three exponential components with lifetimes of 2.50 (sites I and II) and 1.70 and 0.63 ms (sites III and IV). This finding permits the time resolution of the excitation spectrum by determination of the amplitudes of the three components as the excitation wavelength is scanned across the spectral profile in 0.1-nm increments. The amplitudes (intensities at time t = O) are plotted as a function of wavelength and the results fitted to three Lorentzian peaks centered at 579.20, 579.40, and 579.32 nm in order of decreasing lifetimes. In H2O solution only two exponential luminescence decay components are resolvable with lifetimes of 0.41 and 0.27 ms, corresponding to sites I and II and sites III and IV, respectively. These results indicate that two water molecules are coordinated to the Eu3+ ions at sites I and II and at either site III or site IV, with three water molecules at the remaining site.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Circularly polarized luminescence from terbium(III) as a probe of metal ion binding in calcium-binding proteins.

A number of different experimental techniques have been used to probe the details of structural changes on the binding of Ca(II) to the large number of known calcium-binding proteins. The use of luminescent lanthanide(III) ions, especially terbium(III) and europium(III), as substitutional replacement for calcium(II), has led to a number of useful experiments from which important details concerning the metal ion coordination sites have been obtained. This work is concerned with the measurement of the circularly polarized luminescence (CPL) from the 5D4----7F5 transition of Tb(III) bound to the calcium binding sites of bovine trypsin, bovine brain calmodulin, and frog muscle parvalbumin. It is demonstrated that it is possible to make these polarization measurements from very dilute solutions (less than 20 microM) and monitor structural changes as equivalents of Tb(III) are added. It is shown that the two proteins that belong to the class of "EF-hand" structures (calmodulin and parvalbumin) possess quite similar CPL line shapes, whereas Tb(III) bound to trypsin has a much different band structure. CPL results following competitive and consecutive binding of Ca(II) and Tb(III) bound to calmodulin are also reported and yield information concerning known differences between the sequence of binding of these two species.     

Interactions of spin-labeled calmodulin with trifluoperazine and phosphodiesterase in the presence of Ca(II), Cd(II), La(III), Tb(III), and Lu(III)

Bovine calmodulin analogues, spin-labeled at methionine and tyrosine residues, have been utilized in electron paramagnetic resonance (EPR) studies designed to investigate calmodulin interactions with the antipsychotic drug trifluoperazine and the calmodulin-binding protein 3',5'-cyclic nucleotide phosphodiesterase. Trifluoperazine titrations of spin-labeled calmodulin analogues were carried out in the presence of Ca(II), Cd(II), and Tb(III). Similar experiments were performed with the phosphodiesterase in the presence of Ca(II), Cd(II), La(III), Tb(III), and Lu(III). EPR signals from the methionine-directed probe proved to be more sensitive to the binding of target molecules than signals from the tyrosine-directed probe, perhaps indicating that the spin-labeled methionine is at a site close to the target molecule binding site. While the binding of TFP, as monitored by EPR spectral changes in the methionine spin-labeled calmodulin, was in evidence with Ca(II), Cd(II), and all the lanthanides examined, no binding of phosphodiesterase to calmodulin could be detected in the presence of the lanthanide ions, perhaps due to inactivation of the phosphodiesterase by lanthanide ion binding. The abilities of the spin-labeled calmodulins to activate phosphodiesterase were also investigated. The spin-labeled tyrosine calmodulin was able to activate phosphodiesterase as well as native calmodulin, while a lower degree of activation was found when the spin-labeled methionine analogue was used.     

Three Ca2+-binding proteins from porcine liver and intestine differ immunologically and physicochemically and are distinct in Ca2+ affinities

Intestinal brush-border-derived membrane vesicles contain, after demembranation in the presence of Ca2+, a subset of polypeptides that are specifically solubilized by the addition of Ca2+ chelators. As described previously, this fractionation scheme leads to the enrichment of two major proteins (I and II), one of which has been shown to be identical to the cellular p36K target of Rous sarcoma virus-encoded tyrosine-specific protein kinase (Gerke, V., and Weber, K., (1984) EMBO J. 3, 227-233). We have applied a similar protocol to membrane vesicles from porcine liver and purified a third Ca2+-binding protein (III). All three proteins had wide tissue distributions, and were absent from brain, red blood cells, and cardiac and skeletal muscle. Relative amounts varied between tissues, with protein I low in liver and protein III very low in intestine. Despite their similar extractability the three proteins (I, II, and III) are clearly distinct as far as immunological, biochemical, and physicochemical properties are concerned. They also show characteristic differences in their affinities for Ca2+ ions. The association constants of Ca2+ binding for proteins I and III have been estimated by means of indirect methods to be 10(4) M-1 (protein I) and 10(6) M-1 (protein III), while the direct Hummel-Dreyer method reveals Ca2+ binding to protein II, characterized by an association constant of 0.4 X 10(5) M-1 in the absence and 0.2 X 10(5) M-1 in the presence of 2 mM MgCl2. Conformational changes upon binding Ca2+ are described for protein II using circular dichroism, fluorescence emission, and UV difference spectra. These alterations could be attributed to an increased exposure of tyrosine and tryptophan residues to a more aqueous environment, and led to increased hydrophobicity of protein II that would explain the observed Ca2+-dependent interaction with hydrophobic matrices like phenyl-Sepharose.     

High-resolution proton and laser photochemically induced dynamic nuclear polarization NMR studies of cation binding to bovine alpha-lactalbumin

alpha-Lactalbumin (alpha-LA) is a calcium binding protein that also binds Mn(II), lanthanide ions, A1(III), Zn(II), Co(II). The structural implications of cation binding were studied by high-resolution proton (200 MHz) NMR and photochemically induced dynamic nuclear polarization (CIDNP) spectroscopy. Marked changes were observed in the NMR spectra of the apoprotein upon addition of a stoichiometric amount of calcium to yield Ca(II)-alpha-LA, manifested particularly in ring current shifted aliphatic peaks and in several shifts in the aromatic region, all of which were under slow exchange conditions. The CIDNP results showed that two surface-accessible tyrosine residues, assigned as Tyr-18 and -36, became inaccessible to the solvent upon addition of 1:1 Ca(II) to apo-alpha-lactalbumin, while Tyr-103 and Trp-104 remained completely accessible in both conformers. The proton NMR spectra of apo-alpha-LA and A1(III)-alpha-LA were extremely similar, which was also consistent with intrinsic fluorescence results [Murakami, K., & Berliner, L. J. (1983) Biochemistry 22, 3370-3374]. The paramagnetic cation Mn(II) bound to the strong calcium binding site on apo-alpha-LA but also to the weak secondary Ca(II) binding site(s) on Ca(II)-alpha-LA. It was also found that Co(II) bound to some secondary sites on Ca(II)-alpha-LA that overlapped the weak calcium site. All of the lanthanide shift reagents [Pr(III), Eu(III), Tb(III), Dy(III), Tm(III), Yb(III)] bound under slow exchange conditions; their relative affinities for apo-alpha-lactalbumin from competitive binding experiments were Dy(III), Tb(III), and Pr(III) greater than Ca(II) greater than Yb(III).     

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.     

Tb3+ and Ca2+ binding to phosphatidylcholine. A study comparing data from optical, NMR, and infrared spectroscopies.

The paramagnetic and luminescent lanthanides are unique probes of cation-phospholipid interactions. Their spectroscopic properties provide the means to characterize and monitor complexes formed with lipids in ways not possible with biochemically more interesting cations, such as Ca2+. In this work, Tb3+-phosphatidylcholine complexes are described using the luminescence properties of Tb3+, the effect of its paramagnetism on the 31P NMR and 13C NMR spectra of the lipid, and changes in the infrared spectrum of the lipid induced by the cation. There are two Tb3+-phosphatidylcholine complexes with very different coordination environments, as evidenced by changes in the optical excitation spectrum of the lanthanide. The NMR experiments indicate that the two complexes differ in the number of phosphate groups directly coordinating Tb3+. Tb3+ binding induces changes in the phosphodiester infrared bands that are most consistent with bidentate chelation of Tb3+ by each phosphate, whereas Ca2+-induced changes are more consistent with monodentate coordination. The significance of this discrepancy is discussed.     

Estimation of inter-binding-site distances in sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase using Eu(III) luminescence energy transfer

We have used several trivalent lanthanides as probes for the high-affinity Ca(II)-binding site of the Ca(II) + Mg(II)-ATPase of skeletal muscle sarcoplasmic reticulum. The luminescent probes Eu(III) and Tb(III) were excited directly with pulsed laser light and the energy transfer efficiencies to several lanthanide acceptors were measured, under conditions in which most donor-acceptor pair occupied high-affinity Ca(II) sites. We obtain an inter-ionic site distance of about 0.8-0.9 nm. Energy transfer measurements were also done with Eu(III) in at least one Ca(II) site and bidentate Cr-ATP complex at the ATP hydrolytic site. Quenching of Eu(III) luminescence by Cr-ATP was total under these conditions. We calculate an upper limit of 1.0 nm for the distance from the Ca(II) site(s) to the complexed Cr(III) ion at the hydrolytic site.     

Dynamics of Ca2+-saturated calmodulin D129N mutant studied by multiple molecular dynamics simulations

Fifteen independent 1-nsec MD simulations of fully solvated Ca(2+) saturated calmodulin (CaM) mutant D129N were performed from different initial conditions to provide a sufficient statistical basis to gauge the significance of observed dynamical properties. In all MD simulations the four Ca(2+) ions remained in their binding sites, and retained a single water ligand as observed in the crystal structure. The coordination of Ca(2+) ions in EF-hands I, II, and III was sevenfold. In EF-hand IV, which was perturbed by the mutation of a highly conserved Asp129, an anomalous eightfold Ca(2+) coordination was observed. The Ca(2+) binding loop in EF-hand II was observed to dynamically sample conformations related to the Ca(2+)-free form. Repeated MD simulations implicate two well-defined conformations of Ca(2+) binding loop II, whereas similar effect was not observed for loops I, III, and IV. In 8 out of 15 MD simulations Ca(2+) binding loop II adopted an alternative conformation in which the Thr62 >C=O group was displaced from the Ca(2+) coordination by a water molecule, resulting in the Ca(2+) ion ligated by two water molecules. The alternative conformation of the Ca(2+) binding loop II appears related to the "closed" state involved in conformational exchange previously detected by NMR in the N-terminal domain fragment of CaM and the C-terminal domain fragment of the mutant E140Q. MD simulations suggest that conformations involved in microsecond exchange exist partially preformed on the nanosecond time scale.