Probing ribosome structure by europium-induced RNA cleavage

Divalent metal ions are absolutely required for the structure and catalytic activities of ribosomes. They are partly coordinated to highly structured RNA, which therefore possesses high-affinity metal ion binding pockets. As metal ion induced RNA cleavages are useful for characterising metal ion binding sites and RNA structures, we analysed europium (Eu3+) induced specific cleavages in both 16S and 23S rRNA of E. coli. The cleavage sites were identified by primer extension and compared to those previously identified for calcium, lead, magnesium, and manganese ions. Several Eu3+ cleavage sites, mostly those at which a general metal ion binding site had been already identified, were identical to previously described divalent metal ions. Overall, the Eu3+ cleavages are most similar to the Ca2+ cleavage pattern, probably due to a similar ion radius. Interestingly, several cleavage sites which were specific for Eu3+ were located in regions implicated in the binding of tRNA and antibiotics. The binding of erythromycin and chloramphenicol, but not tetracycline and streptomycin, significantly reduced Eu3+ cleavage efficiencies in the peptidyl transferase center. The identification of specific Eu3+ binding sites near the active sites on the ribosome will allow to use the fluorescent properties of europium for probing the environment of metal ion binding pockets at the ribosome's active center.     

Investigating the effects of posttranslational adenylylation on the metal binding sites of Escherichia coli glutamine synthetase using lanthanide luminescence spectroscopy.

Lanthanide luminescence was used to examine the effects of posttranslational adenylylation on the metal binding sites of Escherichia coli glutamine synthetase (GS). These studies revealed the presence of two lanthanide ion binding sites of GS of either adenylylation extrema. Individual emission decay lifetimes were obtained in both H2O and D2O solvent systems, allowing for the determination of the number of water molecules coordinated to each bound Eu3+. The results indicate that there are 4.3 +/- 0.5 and 4.6 +/- 0.5 water molecules coordinated to Eu3+ bound to the n1 site of unadenylylated enzyme, GS0, and fully adenylylated enzyme, GS12, respectively, and that there are 2.6 +/- 0.5 water molecules coordinated to Eu3+ at site n2 for both GS0 and GS12. Energy transfer measurements between the lanthanide donor-acceptor pair Eu3+ and Nd3+, obtained an intermetal distance measurement of 12.1 +/- 1.5 A. Distances between a Tb3+ ion at site n2 and tryptophan residues were also performed with the use of single-tryptophan mutant forms of E. coli GS. The dissociation constant for lanthanide ion binding to site n1 was observed to decrease from Kd = 0.35 +/- 0.09 microM for GS0 to Kd = 0.06 +/- 0.02 microM for GS12. The dissociation constant for lanthanide ion binding to site n2 remained unchanged as a function of adenylylation state; Kd = 3.8 +/- 0.9 microM and Kd = 2.6 +/- 0.7 microM for GS0 and GS12, respectively. Competition experiments indicate that Mn2+ affinity at site n1 decreases as a function of increasing adenylylation state, from Kd = 0.05 +/- 0.02 microM for GS0 to Kd = 0.35 +/- 0.09 microM for GS12. Mn2+ affinity at site n2 remains unchanged (Kd = 5.3 +/- 1.3 microM for GS0 and Kd = 4.0 +/- 1.0 microM for GS12). The observed divalent metal ion affinities, which are affected by the adenylylation state, agrees with other steady-state substrate experiments (Abell LM, Villafranca JJ, 1991, Biochemistry 30:1413-1418), supporting the hypothesis that adenylylation regulates GS by altering substrate and metal ion affinities.     

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.     

Metal substitution in transferrins: specific binding of cerium(IV) revealed by the crystal structure of cerium-substituted human lactoferrin

Proteins of the transferrin family play a key role in iron homeostasis through their extremely strong binding of iron, as Fe3+. They are nevertheless able to bind a surprisingly wide variety of other metal ions. To investigate how metal ions of different size, charge and coordination characteristics are accommodated, we have determined the crystal structure of human lactoferrin (Lf) complexed with Ce4+. The structure, refined at 2.2 A resolution (R=20.2%, Rfree=25.7%) shows that the two Ce4+ ions occupy essentially the same positions as do Fe3+, and that the overall protein structure is unchanged; the same closed structure is formed for Ce2Lf as for Fe2Lf. The larger metal ion is accommodated by small shifts in the protein ligands, made possible by the presence of water molecules adjacent to each binding site. The two Ce4+ sites are equally occupied, indicating that the known difference in the pH-dependent release of Ce4+ arises from a specific protonation event, possibly of the His ligand in one of the binding sites. Comparing the effects of binding Ce4+ with those for the binding of other metal ions, we conclude that the ability of transferrins to accommodate metal ions other than Fe3+ depends on an interplay of charge, size, coordination and geometrical preferences of the bound metal ion. However, it is the ability to accept the six-coordinate, approximately octahedral, site provided by the protein that is of greatest importance.     

Lanthanide spectroscopic studies of the dinuclear and Mg(II)-dependent PvuII restriction endonuclease

Type II restriction enzymes are homodimeric systems that bind four to eight base pair palindromic recognition sequences of DNA and catalyze metal ion-dependent phosphodiester cleavage. While Mg(II) is required for cleavage in these enzymes, in some systems Ca(II) promotes avid substrate binding and sequence discrimination. These properties make them useful model systems for understanding the roles of alkaline earth metal ions in nucleic acid processing. We have previously shown that two Ca(II) ions stimulate DNA binding by PvuII endonuclease and that the trivalent lanthanide ions Tb(III) and Eu(III) support subnanomolar DNA binding in this system. Here we capitalize on this behavior, employing a unique combination of luminescence spectroscopy and DNA binding assays to characterize Ln(III) binding behavior by this enzyme. Upon excitation of tyrosine residues, the emissions of both Tb(III) and Eu(III) are enhanced severalfold. This enhancement is reduced by the addition of a large excess of Ca(II), indicating that these ions bind in the active site. Poor enhancements and affinities in the presence of the active site variant E68A indicate that Glu68 is an important Ln(III) ligand, similar to that observed with Ca(II), Mg(II), and Mn(II). At low micromolar Eu(III) concentrations in the presence of enzyme (10-20 microM), Eu(III) excitation (7)F(0) --> (5)D(0) spectra yield one dominant peak at 579.2 nm. A second, smaller peak at 579.4 nm is apparent at high Eu(III) concentrations (150 microM). Titration data for both Tb(III) and Eu(III) fit well to a two-site model featuring a strong site (K(d) = 1-3 microM) and a much weaker site (K(d) approximately 100-200 microM). Experiments with the E68A variant indicate that the Glu68 side chain is not required for the binding of this second Ln(III) equivalent; however, the dramatic increase in DNA binding affinity around 100 microM Ln(III) for the wild-type enzyme and metal-enhanced substrate affinity for E68A are consistent with functional relevance for this weaker site. This discrimination of sites should make it possible to use lanthanide substitution and lanthanide spectroscopy to probe individual metal ion binding sites, thus adding an important tool to the study of restriction enzyme structure and function.     

Aminoglycoside binding displaces a divalent metal ion in a tRNA-neomycin B complex

Aminoglycosides bind to RNA and interfere with its function, and it has been suggested that aminoglycoside binding to RNA displaces essential divalent metal ions. Here we demonstrate that addition of various aminoglycosides inhibited Pb2+-induced cleavage of yeast tRNA(Phe). Cocrystallization of yeast tRNA(Phe) and an aminoglycoside, neomycin B, resulted in crystals that diffracted to 2.6 A and the structure of the complex was solved by molecular replacement. The structure shows that the neomycin B binding site overlaps with known divalent metal ion binding sites in yeast tRNA(Phe), providing direct evidence for the hypothesis that aminoglycosides displace metal ions. Additionally, the neomycin B binding site overlaps with major determinants for Escherichia coli phenylalanyl-tRNA-synthetase. Here we present data demonstrating that addition of neomycin B inhibited aminoacylation of E. coli tRNA(Phe) in the mid microM range. Given that aminoglycoside and metal ion binding sites overlap, we discuss that aminoglycosides can be considered as 'metal mimics'.     

Probing the metal-binding sites of cod parvalbumin using europium(III) ion luminescence and diffusion-enhanced energy transfer.

Excitation spectroscopy of the 7F0----5D0 transition of Eu3+ and diffusion-enhanced energy transfer are used to study metal-binding characteristics of the calcium-binding protein parvalbumin from codfish. Energy is transferred from Eu3+ ions occupying the CD- and EF-binding sites to the freely-diffusing Co(III) coordination complex energy acceptors: [Co(NH3)6]3+, [Co(NH3)5H2O]3+, [CoF(NH3)5]2+, [CoCl(NH3)5]2+, [Co(NO2)3(NH3)3], and [Co(ox)3]3-. In the absence of these inorganic energy acceptors, the excited-state lifetimes of Eu3+ bound to the CD and EF sites are indistinguishable, even in D2O; however, in the presence of the positively charged energy acceptor complexes, the Eu3+ probes in the cod parvalbumin have different excited-state lifetimes due to a greater energy-transfer site from Eu3+ in the CD site than from this ion in the EF site. The observation of distinct lifetimes for Eu3+ in the two sites allows the study of the relative binding site affinities and selectivity, using other members of the lanthanide ion series. Our results indicate that during the course of a titration of the metal-free protein, Eu3+ fills the two sites simultaneously. Eu3+ is competitively displaced by other Ln3+ ions, with the CD site showing a preference for the larger Ln3+ ions while the EF site shows little, if any, competitive selectivity across the Ln3+ ion series.     

Probing of metal-binding domains of RNA hairpin loops by laser-induced lanthanide(III) luminescence

Direct laser excitation of aqueous Eu(III) bound to specific RNA fragments was used to probe the metal-binding sites of the anticodon loop of tRNA(Phe) from E. coli and of a tetraloop containing a GNRA consensus sequence. Binding of Mg(II) or Eu(III) to either RNA fragment resulted in a higher melting transition, but no global change in structure was observed. Aqueous Eu(III) exhibits a single weak excitation peak at 17273 cm(-1), the intensity of which increased upon addition of the tRNA loop fragment. Analysis of incremental increases in the luminescence intensity upon complexation with the tRNA loop indicated a stoichiometry of one high-affinity Eu(III)-binding site per loop fragment, with a Kd of 1.3 +/- 0.2 microM. Competition experiments between Eu(III) and Mg(II) were consistent with the two metal ions binding to a common site and with an approximately 30-fold lesser affinity of the tRNA loop for Mg(II) than for Eu(III). The rate of luminescence decay following excitation of Eu(III) bound to the tRNA loop corresponded to displacement of up to 4-5 (of a possible 9) waters of hydration on binding to the tRNA loop. By comparison, Eu(III) binds to the DNA analogue of the tRNA loop with an 8-fold lesser affinity and one fewer direct coordination site than to the RNA sequence, suggesting that a 2'OH of RNA is one of the direct ligands. In contrast with the absence of a shift in the excitation peak of aqueous Eu(III) upon formation of the tRNA loop complex, direct excitation of Eu(III) bound to a GNRA tetraloop fragment resulted in a substantially blue-shifted excitation peak (17290 cm(-1)). The tetraloop fragment also has a single Eu(III)-binding site, with a Kd of 12 +/- 3 microM. The bound Eu(III) was competed by Mg(II), although the relative affinity for Mg(II) was approximately 150-450-fold less than that for Eu(III). The Eu(III)-binding site of the tetraloop site is highly dehydrated, with approximately 7 water molecules displaced upon binding by RNA ligands, suggesting that the blue-shift of the excitation peak is the result of Eu(III) specifically bound in a nonpolar site within the GNRA loop structure.     

Quantifying the ion selectivity of the Ca2+ site in photosystem II: evidence for direct involvement of Ca2+ in O2 formation.

Calcium is an essential cofactor in the oxygen-evolving complex (OEC) of photosystem II (PSII). The removal of Ca2+ or its substitution by any metal ion except Sr2+ inhibits oxygen evolution. We used steady-state enzyme kinetics to measure the rate of O2 evolution in PSII samples treated with an extensive series of mono-, di-, and trivalent metal ions in order to determine the basis for the affinity of metal ions for the Ca2+-binding site. Our results show that the Ca2+-binding site in PSII behaves very similarly to the Ca2+-binding sites in other proteins, and we discuss the implications this has for the structure of the site in PSII. Activity measurements as a function of time show that the binding site achieves equilibrium in 4 h for all of the PSII samples investigated. The binding affinities of the metal ions are modulated by the 17 and 23 kDa extrinsic polypeptides; their removal decreases the free energy of binding of the metal ions by 2.5 kcal/mol, but does not significantly change the time required to reach equilibrium. Monovalent ions are effectively excluded from the Ca2+-binding site, exhibiting no inhibition of O2 evolution. Di- and trivalent metal ions with ionic radii similar to that of Ca2+ (0.99 A) bind competitively with Ca2+ and have the highest binding affinity, while smaller metal ions bind more weakly and much larger ones do not bind competitively. This is consistent with a size-selective Ca2+-binding site that has a rigid array of coordinating ligands. Despite the large number of metal ions that competitively replace Ca2+ in the OEC, only Sr2+ is capable of partially restoring activity. Comparing the physical characteristics of the metal ions studied, we identify the pK(a) of the aqua ion as the factor that determines the functional competence of the metal ion. This suggests that Ca2+ is directly involved in the chemistry of water oxidation and is not only a structural cofactor in the OEC. We propose that the role of Ca2+ is to act as a Lewis acid, binding a substrate water molecule and tuning its reactivity.     

Structural characteristics of protein binding sites for calcium and lanthanide ions

Surveys of X-ray structures of Ca2+-containing and lanthanide ion-containing proteins and coordination complexes have been performed and structural features of the metal binding sites compared. A total of 515 structures of Ca2+-containing proteins were considered, although the final data set contained only 44 structures and 60 Ca2+ binding sites with a total of 323 ligands. Eighteen protein structures containing lanthanide ions were considered with a final data set containing eight structures and 11 metal binding sites. Structural features analysed include coordination numbers of the metal ions, the identity of their ligands, the denticity of carboxylate ligands, and the type of secondary structure from which the ligands are derived. Three general types of calcium binding site were identified in the final data set: class I sites supply the Ca2+ ligands from a continuous short sequence of amino acids; class II sites have one ligand supplied by a part of the amino acid sequence far removed from the main binding sequence; and class III sites are created by amino acids remote from one another in the sequence. The abundant EF-hand type of Ca2+ binding site was under-represented in the data set of structures analysed as far as its biological distribution is concerned, but was adequately represented for the chemical survey undertaken. A turn or loop structure was found to provide the bulk of the ligands to Ca2+, but helix and sheet secondary structures are slightly better providers of bidentate carboxylate ligation than turn or loop structures. The average coordination number for Ca2+ was 6.0, though for EF-hand sites it is 7. The average coordination number of a lanthanide ion in an intrinsic protein Ca2+ site was 7.2, but for the adventitious sites was only 4.4. A survey of the Cambridge Structural Database showed there are small-molecule lanthanide complexes with low coordination numbers but it is likely that water molecules, which do not appear in the electron density maps, are present for some lanthanide sites in proteins. A detailed comparison of the well-defined Ca2+ and lanthanide ion binding sites suggests that a reduction of hydrogen bonding associated with the ligating residues of the binding sites containing lanthanide ions may be a response to the additional positive charge of the lanthanide ion. Major structural differences between Ca2+ binding sites with weak and strong binding affinities were not obvious, a consequence of long-range electrostatic interactions and metal ion-induced protein conformational changes modulating affinities.     

Thermodynamics of Ni2+, Cu2+, and Zn2+ binding to the urease metallochaperone UreE

The two Ni2+ ions in the urease active site are delivered by the metallochaperone UreE, whose metal binding properties are central to the assembly of this metallocenter. Isothermal titration calorimetry (ITC) has been used to quantify the stoichiometry, affinity, and thermodynamics of Ni2+, Cu2+, and Zn2+ binding to the well-studied C-terminal truncated H144*UreE from Klebsiella aerogenes, Ni2+ binding to the wild-type K. aerogenes UreE protein, and Ni2+ and Zn2+ binding to the wild-type UreE protein from Bacillus pasteurii. The stoichiometries and affinities obtained by ITC are in good agreement with previous equilibrium dialysis results, after differences in pH and buffer competition are considered, but the concentration of H144*UreE was found to have a significant effect on metal binding stoichiometry. While two metal ions bind to the H144*UreE dimer at concentrations <10 microM, three Ni2+ or Cu2+ ions bind to 25 microM dimeric protein with ITC data indicating sequential formation of Ni/Cu(H144*UreE)4 and then (Ni/Cu)2(H144*UreE)4, or Ni/Cu(H144*UreE)2, followed by the binding of four additional metal ions per tetramer, or two per dimer. The thermodynamics indicate that the latter two metal ions bind at sites corresponding to the two binding sites observed at lower protein concentrations. Ni2+ binding to UreE from K. aerogenes is an enthalpically favored process but an entropically driven process for the B. pasteurii protein, indicating chemically different Ni2+ coordination to the two proteins. A relatively small negative value of DeltaCp is associated with Ni2+ and Cu2+ binding to H144*UreE at low protein concentrations, consistent with binding to surface sites and small changes in the protein structure.     

Lanthanide ion luminescence as a probe of DNA structure. 1. Guanine-containing oligomers and nucleotides.

Laser-induced Eu(3+) luminescence spectroscopy is used to probe the interaction of Eu(3+) ion with guanine-containing nucleotides and single-stranded oligomers. By using time-resolved and non-time-resolved Eu(3+) luminescence techniques, two classes of Eu(3+) binding site are observed in oligo(dG)10, oligo(dG)8, oligo(dG)6, oligo(dG)4, and d-GMP. One class of site binds Eu(3+) ions more strongly than the other. Since the "tight" class of bound Eu(3+) ions have two coordinated water molecules, it is inferred that six or seven atoms from the oligomers are coordinating the Eu(3+). The "weaker" class of Eu(3+) ion sites involve the coordination of six or seven water molecules and therefore, are coordinated by one or two atoms from the oligomer. The tight class of Eu(3+) binding site is attributed to an interstrand association of Eu(3+) with the oligomers forming dimeric or polymeric structures. The dissociation constants (Kd) for the 1:1 complexes Eu(d-GMP)+ and Eu(d-GTP)- have been determined as well as the Kd for the dimerization reaction of Eu(d-GMP)+. The Tb(3+) luminescence enhancement properties of these molecules are also examined in relation to their EU(3+) binding characteristics.     

Conserved aspartic acid 714 in transmembrane segment 8 of the ZntA subgroup of P1B-type ATPases is a metal-binding residue

ZntA from Escherichia coli is a member of the P1B-type ATPase family that confers resistance specifically to Pb2+, Zn2+, and Cd2 salts by active efflux across the cytoplasmic membrane. P1B-type ATPases are important for homeostasis of metal ions such as Cu+, Ag+, Pb2+, Zn2+, Cd2+ Cu2+, and Co2+, with different subgroups showing specificity for different metal ions. Sequence alignments of P1B-type ATPases show that ZntA and close homologues have a strictly conserved Asp714 in the eighth transmembrane domain that is not conserved in other subgroups of P1B-type ATPases. However, in the sarcoplasmic reticulum Ca2+-ATPase, a structurally characterized P-type ATPase, the residue corresponding to Asp714 is a metal-binding residue. Four site-specific mutants at Asp714, D714E, D714H, D714A, and D714P, were characterized. A comparison of their metal-binding affinity with that of wtZntA revealed that Asp714 is a ligand for the metal ion in the transmembrane site. Thus, Asp714 is one of the residues that determine metal ion specificity in ZntA homologues. All four substitutions at Asp714 in ZntA resulted in complete loss of in vivo resistance activity and complete or large reductions in ATPase activity, though D714E and D714H retained the ability to bind metal ions with high affinity at the transmembrane site. Thus, the ability to bind metal ions with high affinity did not correlate with high activity. The metal-binding affinity of the N-terminal site remained unchanged in all four mutants. The affinities of the two metal-binding sites in wtZntA determined in this study are similar to values reported previously for the individual sites in isolated ZntA fragments.     

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)     

Characterization of Ca2+ interactions with matrix metallopeptidase-12: implications for matrix metallopeptidase regulation

Matrix metallopeptidase-12 (MMP-12) binds three calcium ions and a zinc ion, in addition to the catalytic zinc ion. These ions are thought to have a structural role, stabilizing the active conformation of the enzyme. To characterize the importance of Ca2+ binding for MMP-12 activity and the properties of the different Ca2+ sites, the activity as a function of [Ca2+] and the effect of pH was investigated. The enzymatic activity was directly correlated to calcium binding and a Langmuir isotherm for three binding sites described the activity as a function of [Ca2+]. The affinities for two of the binding sites were quantified at several pH values. At pH 7.5, the KD was 0.1 mM for the high-affinity binding site, 5 mM for the intermediate-affinity binding site and >100 mM for the low-affinity binding site. For all three sites, the affinity for calcium decreased with reduced pH, in accordance with the loss of interactions upon protonation of the calcium-co-ordinating aspartate and glutamate carboxylates at acidic pH. The pKa values of the calcium binding sites with the highest and intermediate affinities were determined to be 4.3 and 6.5 respectively. Optimal pH for catalysis was above 7.5. The low-, intermediate- and high-affinity binding sites were assigned on the basis of analysis of three-dimensional-structures of MMP-12. The strong correlation between MMP-12 activity and calcium binding for the physiologically relevant [Ca2+] and pH ranges studied suggest that Ca2+ may be involved in controlling the activity of MMP-12.     

Ni2+-binding RNA motifs with an asymmetric purine-rich internal loop and a G-A base pair.

RNA molecules with high affinity for immobilized Ni2+ were isolated from an RNA pool with 50 randomized positions by in vitro selection-amplification. The selected RNAs preferentially bind Ni2+ and Co2+ over other cations from first series transition metals. Conserved structure motifs, comprising about 15 nt, were identified that are likely to represent the Ni2+ binding sites. Two conserved motifs contain an asymmetric purine-rich internal loop and probably a mismatch G-A base pair. The structure of one of these motifs was studied with proton NMR spectroscopy and formation of the G-A pair at the junction of helix and internal loop was demonstrated. Using Ni2+ as a paramagnetic probe, a divalent metal ion binding site near this G-A base pair was identified. Ni2+ ions bound to this motif exert a specific stabilization effect. We propose that small asymmetric purine-rich loops that contain a G-A interaction may represent a divalent metal ion binding site in RNA.     

Proton nuclear magnetic resonance and electron paramagnetic resonance studies on skeletal muscle actin indicate that the metal and nucleotide binding sites are separate

The distance separating the high-affinity binding sites of actin for a divalent metal ion and nucleotide was evaluated by using high-resolution proton NMR and EPR spectroscopy. Replacement of the Ca2+ or Mg2+ bound to the high-affinity divalent cation site of G-actin by trivalent lanthanide ions such as La3+, EU3+, or Gd3+ results in an increase in the mobility of the bound ATP as observed in the NMR spectra of G-actin monomers. Little difference was observed between the spectra obtained in the presence of the diamagnetic La3+ control and the paramagnetic ions Eu3+ and Gd3+ which respectively shift and broaden the proton resonances of amino acids in the vicinity of the binding site. Analysis of the NMR spectra indicates that the metal and nucleotide binding sites are separated by a distance of at least 16 A. In the past, the metal and ATP have been widely assumed to bind as a complex. Further verification that the two sites on actin are physically separated was obtained by using an ATP analogue with a nitroxide spin-label bound at the 6' position of the purine ring. An estimate of the distance was made between the site containing the ATP analogue and the paramagnetic ion, Mn2+, bound to the cation binding site. These EPR experiments were not affected by the state of polymerization of the actin. The data obtained by using this technique support the conclusion stated above, namely, that the cation and nucleotide sites on either G- or F-actin are well separated.     

Binding of europium(III) ions to RNA stem loops: role of the primary hydration sphere in complex formation

Understanding the process by which RNA molecules fold into stable structures includes study of the role of site-bound metal ions. Because the alkaline earth metal ions typically associated with RNA structure [most often Mg(II)] do not provide convenient spectroscopic signals, replacement with metal ions having spectroscopically useful properties has been a valuable approach. The luminescence properties of the lanthanide(III) series, in particular europium(III), have made them useful in the study of complexation with biomolecules. We review the physical, chemical, and spectroscopic characteristics of Eu(III) that contribute to its value as a probe of RNA-metal ion interactions, and examples of information obtained from studies of Eu(III) bound to small RNA stem loops. Although Eu(III) has similar site preference to Mg(II), luminescence and isothermal titration calorimetry measurements indicate that Ln(III) loses water molecules from the inner hydration sphere more readily than does Mg(II), resulting in more direct coordination between RNA and the metal ion and very different energetics of binding. In some cases, e.g., a GAAA tetraloop, binding appears to occur by a lock and key process; in the same base sequence containing certain deoxynucleoside substitutions that alter loop structure, binding appears to occur by an induced fit process.     

Binding of Eu3+ to cardiac sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase-laser excited Eu3+ spectroscopic studies.

The binding of Eu3+ with Ca2+-stimulated, Mg2+-dependent adenosine triphosphatase ([Ca2+ + Mg2+]-ATPase) of cardiac sarcoplasmic reticulum (SR) has been investigated using direct laser excited Eu3+ luminescence. Eu3+ is found to inhibit both Ca2+-dependent ATPase activity and Ca2+-uptake in a parallel manner. This is attributed to the binding of Eu3+ to the high affinity Ca2+-binding sites. The Ki for Ca2+-dependent ATPase is approximately 50 nM. The 7F0----5D0 excitation spectrum of Eu3+ in cardiac SR shows a peak at 579.3 nm, as compared to 578.8 nm in potassium-morpholino propane sulfonic acid (K-MOPS) pH 6.8. Upon binding with cardiac SR, Eu3+ shows an increase in fluorescence intensity as well as in lifetime values. The fluorescence decay of bound Eu3+ exhibits a double-exponential curve. The apparent number of water molecules in the first coordination sphere of Eu3+ in SR is 2.8 for the short component and 1.0 for the long component. In the presence of ATP, a further increase in fluorescence lifetimes is observed, and the number of water molecules in the first coordination sphere of Eu3+ is reduced further to 1.3 and 0.5. The double exponential nature of the decay curve and the different number of water molecules coordinated to Eu3+ for both decay components suggest that Eu3+ binds to two sites and that these are heterogeneous. The reduction in the number of H2O ligands in the presence of ATP shows a change in the molecular environment of the Eu3+-binding sites upon phosphoenzyme formation, with a movement of Eu3+ to an occluded site on the enzyme.     

Metal induced structural changes observed in hexameric insulin

The metal ions in insulin hexamer play a crucial role in the T to R conformational transitions. We have determined the crystal structures of 2Mn2+, 1Rb1+ and 4Ni2+ human arg-insulin and compared them with the 2Zn2+ structure. The first two structures exist in the T3R3f state like the native 2Zn2+ arg-insulin, while the 4Ni2+ adopts a T6 conformation. The metal coordination is found to be tetrahedral in all the structures except that of nickel where a dual octahedral and tetrahedral coordination is found at one site. Rubidium occupies only one of the high affinity metal binding sites. The metal induced structural changes observed, have been explained.