Evidence of a calcium-induced structural change in the ATP-binding site of the sarcoplasmic-reticulum Ca2+-ATPase using terbium formycin triphosphate as an analogue of Mg-ATP

Terbium ions and terbium formycin triphosphate have been used to investigate the interactions between the cation and nucleotide binding sites of the sarcoplasmic reticulum Ca2+-ATPase. Three classes of Tb3+-binding sites have been found: a first class of low-affinity (Kd = 10 microM) corresponds to magnesium binding sites, located near a tryptophan residue of the protein; a second class of much higher affinity (less than 0.1 microM) corresponds to the calcium transport sites, their occupancy by terbium induces the E1 to E2 conformational change of the Ca2+-ATPase; a third class of sites is revealed by following the fluorescence transfer from formycin triphosphate (FTP) to terbium, evidencing that terbium ions can also bind into the nucleotide binding site at the same time as FTP. Substitution of H2O by D2O shows that Tb-FTP binding to the enzyme nucleotide site is associated with an important dehydration of the terbium ions associated with FTP. Two terbium ions, at least, bind to the Ca2+-ATPase in the close vicinity of FTP when this nucleotide is bound to the ATPase nucleotide site. Addition of calcium quenches the fluorescence signal of the terbium-FTP complex bound to the enzyme. Calcium concentration dependence shows that this effect is associated with the replacement of terbium by calcium in the transport sites, inducing the E2----E1 transconformation when calcium is bound. One interpretation of this fluorescence quenching is that the E1----E2 transition induces an important structural change in the nucleotide site. Another interpretation is that the high-affinity calcium sites are located very close to the Tb-FTP complex bound to the nucleotide site.     

Terbium ion binding to Limuluspolyphemus hemocyanin

Terbium ion binds to calcium-free $\text{Limulus}$ hemocyanin at pH 7.0 and 8.9, and promotes the aggregation of hemocyanin subunits, a phenomenon associated with calcium binding. An excitation maximum for the bound terbium at 293 nm and the results of treating the hemocyanin with N-bromosuccinimide indicate that energy transfer from tryptophan to the bound terbium is responsible for the enhancement of terbium fluorescence. At pH 8.9, addition of calcium to hemocyanin containing bound terbium results in only a partial loss of terbium fluorescence, suggesting heterogeneity in the terbium binding sites. Titration of hemocyanin with terbium also indicates multiple binding sites.     

Interaction of terbium and calcium with chicken cystatin

The emission intensity of the fluorescent lanthanide, terbium, is shown to be enhanced upon binding to chicken cystatin. Fluorescence titrations indicate the presence of a single high affinity binding site per molecule. Binding of the terbium results in a 29% quenching of the fluorescence of the single tryptophan residue in the molecule. Calcium displaces the terbium from cystatin as judged by the decrease of terbium fluorescence in competition titrations. Similar titrations with magnesium or strontium demonstrate that the metal binding site of cystatin exhibits specificity for calcium or terbium. Analysis of the N-terminal sequence of chicken cystatin suggests the presence of a putative consensus sequence for a metal binding site between residues 13 and 24. Calcium causes a 17% decrease in the tryptophan fluorescence of cystatin, indicating that an induced conformational change accompanies metal binding. The increased quenching observed with terbium appears to be the result of resonance energy transfer from tryptophan to terbium. From the critical distance for energy transfer from tryptophan to terbium, it is estimated that the terbium binding site lies approximately 12 A from the single tryptophan residue in the molecule.     

Binding of terbium(III) to yeast enolase

Several independent criteria indicate 2 mol of terbium(III) bind to yeast enolase in the absence of substrate-fluorescence titrations of enzyme and metal, effects on thermal stability and published ultrafiltration and inhibition experiments. These measurements also suggest the terbium binding sites are the same as those normally occupied by “conformational” magnesium. Terbium binds much more strongly than magnesium, however, and measurements of the kinetics of the absorbance change in the terbium-enzyme on adding excess EDTA suggest the terbium-enzyme dissociation constant is about $\text{1}\text{500}$ that of the magnesium-enzyme. Measurements of enzyme activity as a function of substrate concentration show that terbium permits no enzymatic activity. However, magnesium competes more effectively with the lanthanide if the substrate analogue 3-aminoenolpyruvate 2-phosphate (AEP) is present.The fluorescence of the lanthanide is not readily observed on exciting the terbium-enzyme at 280 nm, indicating the absence of tyrosines or tryptophans in the coordination sphere of the metal. Excitation of terbium using 488 nm radiation from an argon ion laser shows the fluorescence of the metal is enhanced by binding to the enzyme. EDTA and carbonate have similar effects. This suggests carboxyl groups are involved in binding metal at the conformational sites of yeast enolase. Measurements of lifetimes of enzyme-bound terbium in the presence and absence of D₂O indicated three moles of water remained on each of the bound metals, independently of the buffer used. If enzyme-bound terbium is assumed to be nine-coordinate, the metal must bind to six groups from the enzyme. The presence of substrate does not markedly affect the emission spectrum of the bound terbium or the number of water molecules remaining on the metal, but calorimetric measurements show that substrate binds to the terbium enzyme.     

The location of spermine in bacterial ribosomes as indicated by 1,5-difluoro-2,4-dinitrobenzene and by ethidium bromide

1. When the binding of ethidium bromide to rRNA is measured both in the presence and in the absence of spermine, by spectrophotometric titrations, by gel filtration, or by the changes in fluorescence intensity, spermine competes with ethidium bromide for sites on the rRNA; under the conditions used in these experiments ethidium bromide is bound to the double-stranded regions of rRNA. 2. When an excess of ethidium bromide is added to ribosomes from Bacillus stearothermophilus approx. 80% of the endogenous spermine is displaced from the ribosomes. 3. [(14)C]Spermine is fixed to ribosomes by either formaldehyde or 1,5-difluoro-2,4-dinitrobenzene. Most of the [(14)C]spermine, fixed to ribosomes by 1,5-difluoro-2,4-dinitrobenzene, attaches to the ribosomal protein. 4. It is concluded that most of the endogenous spermine is bound to the double-stranded RNA in ribosomes, and that some of these double-stranded regions to which spermine is attached also have ribosomal proteins bound to them.     

The binding sites for tRNA on eukaryotic ribosomes.

We have studied the non-enzymic binding of phe-tRNA to ribosomes from rat liver using deacylated tRNA to inhibit binding to the P-site and puromycin (5 x 10-minus3M) to inhibit binding to the A-site. We conclude that at a low concentration of magnesium ions (10mM) phe-tRNA is bound only at the A-site of 80S irbosomes, whereas at a high concentration of magnesium ions (40mM) phe-tRNA is also bound at the P-site. Studies with edeine indicate that, during non-enzymic binding of phe-tRNA, eukaryotic ribosomes (in contrast to prokarotic ribosomes) have the A-site of the 60S subunit and the initiation site of the 40S subunit juxtaposed. This may account for the differences observed, in formation of diphenylalanyl-tRNA and phenylalanyl-puromycin, between phe-tRNA bound non-enzymically to the P-sites of eukaryotic and prokaryotic ribosomes.     

Conformational studies of Escherichia coli ribosomes with the use of acridine orange as a probe

The interaction of E. coli vacant ribosomes with acridine orange (AO) was studied, to obtain conformational information about rRNAs in ribosomes. Acridine orange binds to an RNA in two different modes: cooperative outside binding with stacking of bound AO's and intercalation between nucleotide bases. Free 16S and 23S rRNAs have almost identical affinities to AO. At 1 mM Mg2+, AO can achieve stacking binding on about 40% of rRNA phosphate groups. The number of stacking binding sites falls to about 1/3 in the 30S subunit in comparison with free 16S rRNA. In the 50S subunit, the number of stacking binding sites is only 1/5 in comparison with free 23S rRNA. Mg2+ ions are more inhibitory for the binding of AO to ribosomes than to free rRNAs. The strength of stacking binding appears to be more markedly reduced by Mg2+ in active ribosomes than in rRNAs. "Tight couple" 70S particles are less accessible for stacking binding than free subunits. The 30S subunits that have irreversibly lost the capability for 70S formation under low Mg2+ conditions have an affinity to AO that is very different from that of active 30S but similar to that of free rRNA, though the number of stacking binding sites is little changed by the inactivation. 70S and 30S ribosomes with stacking bound AO's have normal sedimentation constants, but the 50S subunits reversibly form aggregates.     

Interaction of thiostrepton and elongation factor-G with the ribosomal protein L11-binding domain

Ribosomal protein L11 and the L11 binding region of ribosomal RNA constitute an important domain involved in active functions of the ribosome during translation. We studied the effects of L11 knock-out and truncation mutations on the structure of the rRNA in this region and on its interactions with a translation elongation factor and the antibiotic thiostrepton. The results indicated that the structure of the L11-binding rRNA becomes conformationally flexible when ribosomes lack the entire L11 protein, but not when the C-terminal domain is present on ribosomes. Probing wild type and mutant ribosomes in the presence of the antibiotic thiostrepton and elongation factor-G (EF-G) rigorously localized the binding cleft of thiostrepton and suggested a role for the rRNA in the L11-binding domain in modulating factor binding. Our results also provide evidence that the structure of the rRNA stabilized by the C-terminal domain of L11 is necessary to stabilize EF-G binding in the post-translocation state, and thiostrepton may modulate this structure in a manner that interferes with the ribosome-EF-G interaction. The implications for recent models of thiostrepton activity and factor interactions are discussed.     

Evidence for RNA in the peptidyl transferase center of Escherichia coli ribosomes as indicated by fluorescence.

A coumarin derivative was covalently attached to either the amino acid or the 5' end of phenylalanine-specific transfer RNA (tRNA(phe)). Its fluorescence was quenched by methyl viologen when the tRNA was free in solution or bound to Escherichia coli ribosomes. Methyl viologen as a cation in solution has a strong affinity for the ionized phosphates of a nucleic acid and so can be used to qualitatively measure the presence of RNA in the immediate vicinity of the tRNA-linked coumarins upon binding to ribosomes. Fluorescence lifetime measurements indicate that the increase in fluorescence quenching observed when the tRNAs are bound into the peptidyl site of ribosomes is due to static quenching by methyl viologen bound to RNA in the immediate vicinity of the fluorophore. The data lead to the conclusion that the ribosome peptidyl transferase center is rich in ribosomal RNA. Movement of the fluorophore at the N-terminus of the nascent peptide as it is extended or movement of the tRNA acceptor stem away from the peptidyl transferase center during peptide bond formation appears to result in movement of the probe into a region containing less rRNA.     

Binding of magnesium ions and ethidium bromide: comparison of ribosomes and free ribosomal RNA.

Comparative studies of free ribosomal RNA and ribosomes were made with two probes, Mg++ ions and ethidium bromide, which interact with RNA in different ways. Mg++. E. coli 16 S rRNA and 30 S ribosomes were equilibrated with four different buffers. Equilibration required several days at 4 degrees and several hours at 37 degrees. In all buffers ribosomes bound more Mg than free rRNA, the difference sometimes reaching 20--30%. Ribosomes were more resistant than free rRNA to heat denaturation and their denaturation was more highly cooperative. Ribosomes that bound more Mg++ had higher denaturation temperatures. Ethidium bromide. Fluorescence enhancement studies of ethidium intercalation showed the free 16 S rRNA to have 50--80 binding sites per molecule. A large fraction of these sites were present and accessible in the ribosome, but their ethidium-binding constants were reduced by an order of magnitude. In addition, free rRNA contained a small number of very strong binding sites that were virtually absent in the ribosomes.     

Magnesium binding to yeast ribosomes

This paper describes a theoretical and experimental analysis of the binding of magnesium ions to yeast, ribosomes. In the theoretical considerations the interactions between charges located on a macroion are included. In the calculations these interactions result in a term, in which both the charge and the radius of the macroion are accounted for. It appears that on dissociation of the ribosomes both the charge and the radius change, but in such a way, that the term, which accounts for the electrostatic interactions, remains constant. As a consequence the dissociation can lie neglected in the analyses of the binding experiments. Our experiments indicate that two binding reactions between ribosomes and magnesium ions occur. The endpoints of these reactions correspond to about 0.40 and 1.0 equivalent magnesium per ribosomal phosphate, respectively. The pK values are about 3.8 and 2.2, respectively. The experimental results indicate that the effect, of monovalent cations can be explained as a pure ionic strength effect, though the binding of monovalent cations could not be excluded completely.     

Structural analysis of the peptidyl transferase region in ribosomal RNA of the eukaryote Xenopus laevis

Accessible single-strand bases in Xenopus laevis 28 S ribosomal RNA (rRNA) Domain V, the peptidyl transferase region, were determined by chemical modification with dimethylsulfate, 1-cyclohexyl-3-(2-morpholinoethyl-carbodiimide metho-p-toluene sulfonate and kethoxal, followed by primer extension. The relative accessibilities of three rRNA substrates were compared: deproteinized 28 S rRNA under non-denaturing conditions (free 28 S rRNA), 60 S subunits and 80 S ribosomes. Overall, our experimental results support the theoretical secondary structure model of Domain V derived by comparative sequence analysis and compensatory base-pair changes, and support some theoretical tertiary interactions previously suggested by covariation. The 60 S subunits and 80 S ribosomes generally show increasing resistance to chemical modification. Bases which are sensitive in free 28 S rRNA but protected in 60 S subunits may be sites for ribosomal protein binding or induced structural rearrangements. Another class of nucleotides is distinguished by its sensitivity in 60 S subunits but protection in 80 S ribosomes; these nucleotides may be involved in subunit-subunit interactions or located at the interface of the ribosome. We found a third class of bases, which is protected in free 28 S rRNA but sensitive in 60 S subunits and/or 80 S ribosomes, suggesting that structural changes occur in Domain V as a result of subunit assembly and ribosome formation. One such region is uniquely hypersensitive in eukaryotic ribosomes but is absent in Escherichia coli ribosomes. Sites that we determined to be accessible on empty 80 S ribosomes could serve as recognition sites for translation components.     

Functional characterization of yeast mitochondrial release factor 1

The yeast Saccharomyces cerevisiae mitochondrial release factor was expressed from the cloned MRF1 gene, purified from inclusion bodies, and refolded to give functional activity. The gene encoded a factor with release activity that recognized cognate stop codons in a termination assay with mitochondrial ribosomes and in an assay with Escherichia coli ribosomes. The noncognate stop codon, UGA, encoding tryptophan in mitochondria, was recognized weakly in the heterologous assay. The mitochondrial release factor 1 protein bound to bacterial ribosomes and formed a cross-link with the stop codon within a mRNA bound in a termination complex. The affinity was strongly dependent on the identity of stop signal. Two alleles of MRF1 that contained point mutations in a release factor 1 specific region of the primary structure and that in vivo compensated for mutations in the decoding site rRNA of mitochondrial ribosomes were cloned, and the expressed proteins were purified and refolded. The variant proteins showed impaired binding to the ribosome compared with mitochondrial release factor 1. This structural region in release factors is likely to be involved in codon-dependent specific ribosomal interactions.     

Activation of calpain I and calpain II: a comparative study using terbium as a fluorescent probe for calcium-binding sites

The present study demonstrates the activation of calpain I and calpain II by micromolar levels of terbium and has utilized the enhancement in the fluorescence of protein-bound terbium to study and compare the calcium binding sites of the two enzymes. Calpain I and calpain II were isolated from bovine erythrocytes and brain, respectively. While the rates of activation of calpain I by terbium and calcium are comparable, the rate of activation of calpain II was much greater in the presence of terbium than in the presence of calcium. Binding of terbium ions to calpains was monitored by the enhanced terbium fluorescence and by the changes in the intrinsic protein fluorescence of calpains. Stoichiometric titrations indicated that calpain I and calpain II bound four and six molar equivalents of terbium ion, respectively. During the titration, the intrinsic protein fluorescence of calpain II was successively quenched whereas that of calpain I showed an abrupt drop just prior to the saturation. The association constants (Ka) increased from 10(5) to 10(7) M-1 for calpain I and from 10(4) to 10(6) M-1 for calpain II with addition of increasing molar equivalents of terbium. Titration of enzymatic activities with calcium showed that the activation of calpain I required fewer molar equivalents of metal ions than were necessary for the activation of calpain II, in agreement with stoichiometric titration with terbium.     

Observations on the binding of lanthanides and calcium to vitamin D-dependent chick intestinal calcium-binding protein. Implications regarding calcium-binding protein function

The binding of calcium and terbium to purified chick vitamin D-dependent intestinal calcium-binding protein was studied by terbium fluorescence, circular dichroism, and intrinsic protein fluorescence techniques. Calcium-binding protein bound, with high affinity, at least 3 mol of terbium/mol of protein; numerous low affinity terbium-binding sites were also noted. The three highest affinity sites were resolved into one very high affinity site (site A) and two other sites (sites B and C) with slightly lower affinity. Resonance energy transfer from tryptophan residues to terbium occurred only with site A. This site was filled before sites B and C. Competition experiments in which calcium was used to displace terbium bound to the protein showed that larger amounts of calcium were needed to displace terbium from site A than from sites B and C. Energy transfer from terbium to holmium indicated that the terbium-binding sites (B and C) were located close to each other (about 7-12 A) but were distant (greater than 12 A) from site A. The addition of EDTA to calcium-binding protein resulted in a 25% decrease in intrinsic protein fluorescence, suggesting a conformational change in the protein. The titration of EDTA-treated calcium-binding protein with calcium resulted in recovery of intrinsic protein fluorescence. A reversible calcium-dependent change in the ellipticity of calcium-binding protein in circular dichroism experiments was also seen. These observed properties suggest that vitamin D-dependent chick intestinal calcium-binding protein behaves in a manner similar to other well-known calcium-binding regulatory proteins.     

Interaction between the erythromycin and chloramphenicol binding sites on the Escherichica coli ribosome.

The effects of chloramphenical on the binding kinetics of a fluorescein isothiocyanate derivative of 9(S)-erythromycylamine with 70S and 50S ribosomes have been studied by direct fluorimetric measurements. While chloramphenicol had little effect on the second-order 70S binding rate of the erythromycin analogue, it substantially reduced the dissociation rate of the fluorescent antibiotic-70S ribosome complex. This could be explained by simultaneous binding of both antibiotics to the 70S ribosome. The kinetic results suggest that chloramphenicol-saturated 70S particles bind the erythromycin analogue four times stronger and this was confirmed by direct binding studies. In additon, chloramphenicol causes a twofold increase in the intrinsic fluorescence of the 70S-bound analogue. This increase in fluorescence was used to study the kinetics of chloramphenicol binding to 70S ribosomes containing the fluorescent derivative. The fluorescence change followed first-order kinetics, suggesting that chloramphenicol induces a conformational change in the 70S particle. This could explain both its effect on erythromycin binding and on the fluorescence of bound analogue. Less detailed results with the 50S particle indicate a qualitively similar picture of erythromycin-chloramphenicol interactions.     

Association of a group I intron with its splice junction in 50S ribosomes: implications for intron toxicity.

The effect of genetic context on splicing of group I introns is not well understood at present. The influence of ribosomal RNA conformation on splicing of rDNA introns in vivo was investigated using a heterologous system in which the Tetrahymena group I intron is inserted into the homologous position of the Escherichia coli 23S rRNA. Mutations that block splicing in E. coli result in accumulation of unspliced 23S rRNA that is assembled into 50S complexes, but not 70S ribosomes. The data indicate that accommodation of the intron structure on the surface of the 50S subunit inhibits interactions with the small ribosomal subunit. Spliced intron RNA also remains noncovalently bound to 50S subunits on sucrose gradients. This interaction appears to be mediated by base pairing between the intron guide sequence and the 23S rRNA, because the fraction of bound intron RNA is reduced by point mutations in the IGS or deletion of the P1 helix. Association of the intron with 50S subunits correlates with slow cell growth. The results suggest that group I introns have the potential to inhibit protein synthesis in prokaryotes by direct interactions with ribosomes.     

Proflavine binding to yeast rRNA and ribosomes as related to structure

Proflavine binding experiments were carried out with yeast rRNA, native and “unfolded” ribosomes; the binding constants and the number of binding sites were calculated by a spectroscopic method. The study of the intercalation complexes by fluorescence and electric dichroism shows the intercalation binding sites to involve two subtypes of sites, which could be related to different nucleotide composition and secondary structure of the rRNA regions, i.e., binding sites located in the (A + U)-rich single strands and binding sites located in the (G + C)-rich double-helical strands (fluorescence quenching sites). Electric dichroism of complexed proflavine is interpreted in terms of rRNA conformation within the ribosomes. The conclusions are in agreement with the ribosomal model of Cox and Bonanou and show that, according to this model, the base planes of the nucleotides are not all parallel in the native ribsome, but rather radiate around the folding axis of the ribonucleoprotein sheet.     

Synthesis,characterization, and binding assessment with human serum albumin of three bipyridine lanthanide(III) complexes

In this work, the terbium(III), dysprosium(III), and ytterbium(III) complexes containing 2, 2′-bipyridine (bpy) ligand have been synthesized and characterized using CHN elemental analysis, FT-IR, UV–Vis and ¹H-NMR techniques and their binding behavior with human serum albumin (HSA) was studied by UV–Vis, fluorescence and molecular docking examinations. The experimental data indicated that all three lanthanide complexes have high binding affinity to HSA with effective quenching of HSA fluorescence via static mechanism. The binding parameters, the type of interaction, the value of resonance energy transfer, and the binding distance between complexes and HSA were estimated from the analysis of fluorescence measurements and Förster theory. The thermodynamic parameters suggested that van der Waals interactions and hydrogen bonds play an important role in the binding mechanism. While, the energy transfer from HSA molecules to all these complexes occurs with high probability, the order of binding constants (BpyTb > BpyDy > BpyYb) represents the importance of radius of Ln³⁺ ion in the complex-HSA interaction. The results of molecular docking calculation and competitive experiments assessed site 3 of HSA, located in subdomain IB, as the most probable binding site for these ligands and also indicated the microenvironment residues around the bound mentioned complexes. The computational results kept in good agreement with experimental data.     

Interaction of unfolded tRNA with the 3''-terminal region of E. coli 16S ribosomal RNA.

Fragments of tRNA possessing a free TpsiC-loop or a free D-loop form stable complexes with the colicin fragment (1494-1542) of 16S ribosomal RNA from E. coli. The colicin fragment does not bind to tRNA in which the T-loop and the D-loop are involved in tertiary interactions. Colicin cleavage of the 16S rRNA from E. coli is inhibited by aminoacyl-tRNA or tRNA fragments, indicating that a similar interaction may take place on the intact 70S ribosomes. The oligonucleotide d(G-T-T-C-G-A)homologous to the conserved sequence G-T-psi-C-Pu-(m1)A in the TpsiC-region of many elongator tRNAs binds to the conserved sequence U-C-G-mU-A-A-C (1495-1501) of the 16S rRNA. It is suggested that the 3'-end of the 16S rRNA may provide the part of the binding site for the elongator tRNAs on bacterial ribosomes.