Interactions of porphyrins and transfer RNA

The interactions of the free base porphyrin, tetra-(4N-methylpyridyl)porphyrin and its copper(II), manganese(III) and zinc(II) complexes with brewer's yeast type V phenylalaninyl tRNA were evaluated by UV-visible spectroscopy, circular dichroism and melting temperature studies over a range of magnesium ion concentrations and ionic strengths. Scatchard analysis of absorption spectra of the porphyrins in the presence of tRNA showed the free base, copper and zinc porphyrins to have binding constants of 7.3 X 10(7), 1.7 X 10(6) and 2.3 X 10(8), respectively; the manganese(III) complex did not demonstrate changes in its electronic spectra that enable the calculation of a binding constant. The results of the spectroscopic studies indicate a mode of binding for the free base, copper(II) and zinc(II) complexes that is neither intercalative nor simply outside electrostatic. The magnitude of the binding constants and the UV-visible results support intercalation, but the analyses of the thermal denaturation studies and the circular dichroism evaluations suggest that the porphyrins are associating at a single site in a fold of the tertiary structure of the tRNA close to several crucial hydrogen bonds, perhaps in the vicinity of the P10 loop. That the manganese(III) complex does not bind in this site points to constraints on the axial thickness of a molecule that may be accommodated in this locus.     

Binding of oxytocin and 8-arginine-vasopressin to neurophysin studied by 15N NMR using magnetization transfer and indirect detection via protons

NMR was used to monitor the binding to neurophysin of oxytocin and 8-arginine-vasopressin, 15N labeling being used to identify specific backbone 15N and 1H signals. The most significant effects of binding were large downfield shifts in the amino nitrogen resonance of Phe-3 of vasopressin and in its associated proton, providing evidence that the peptide bond between residues 2 and 3 of the hormones is hydrogen-bonded to the protein within hormone-neurophysin complexes. Suggestive evidence of hydrogen bonding of the amino nitrogen of Tyr-2 was also obtained in the form of decreased proton exchange rates on binding; however, the chemical shift changes of this nitrogen and its associated proton indicated that such hydrogen bonding, if present, is probably weak. Shifts in the amino nitrogen of Asn-5 and in the -NH protons of both Asn-5 and Cys-6 demonstrated that these residues are significantly perturbed by binding, suggesting conformational changes of the ring on binding and/or the presence of binding sites on the hormone outside the 1-3 region. No support was obtained for the thesis that there is a significant second binding site for vasopressin on each neurophysin chain. The behavior of both oxytocin and vasopressin on binding was consistent with formation of 1:1 complexes in slow exchange with the free state under most pH conditions. At low pH there was evidence of an increased exchange rate. Additionally, broadening of 15N resonances in the bound state at low pH occurred without a corresponding change in the resonances of equilibrating free hormone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spectroscopic studies on the copper(II) complexes of carnosine

Carnosine complexes with copper(II) ions were studied with magnetic resonance techniques over a wide range of ligand to metal ratios at various pH values. Water proton relaxation rates increased with decreasing carnosine to copper ratios until a molar ratio of 48 was reached. Over the ratio range of 48–2 carnosine molecules per copper ion, the relaxation rate decreased so that in the 2:1 carnosine-copper(II) complex, the water-copper(II) distance was estimated to be 1.92 Å. Proton NMR studies revealed the broadening of imidazole proton lines at high mole ratios followed by other histidyl protons as the ratio decreased. The β-alanyl methylene protons were the last to be broadened by the addition of copper(II) ions. Carbon to copper(II) distances were determined for the carnosine to copper mole ratios of 500:1 and 5000:1. EPR spectra obtained at 93°K revealed the probable existence of four carnosine imidazoles as the sole coordinated ligands to copper(II) at high dipeptide-to-metal ratios (>10). At mole ratios below four, nuclear hyperfine lines characteristic of both monomeric and dimeric carnosine-copper(II) forms were observed. These results reveal that imidazole from carnosine is the sole ligand contributed to copper(II) for coordination over the pH range 5 to 7 at high carnosine to copper(II) ratios     

Copper(II) complexes of carnosine, glycylglycine, and glycylglycine-imidazole mixtures

Carnosine complexes with copper(II) ions were studied with magnetic resonance techniques over a wide range of ligand to metal ratios at various pH values. Water proton relaxation rates increased with decreasing carnosine to copper ratios until a molar ratio of 48 was reached. Over the ratio range of 48–2 carnosine molecules per copper ion, the relaxation rate decreased so that in the 2:1 carnosine-copper(II) complex, the water-copper(II) distance was estimated to be 1.92 Å. Proton NMR studies revealed the broadening of imidazole proton lines at high mole ratios followed by other histidyl protons as the ratio decreased. The β-alanyl methylene protons were the last to be broadened by the addition of copper(II) ions. Carbon to copper(II) distances were determined for the carnosine to copper mole ratios of 500:1 and 5000:1. EPR spectra obtained at 93°K revealed the probable existence of four carnosine imidazoles as the sole coordinated ligands to copper(II) at high dipeptide-to-metal ratios (>10). At mole ratios below four, nuclear hyperfine lines characteristic of both monomeric and dimeric carnosine-copper(II) forms were observed. These results reveal that imidazole from carnosine is the sole ligand contributed to copper(II) for coordination over the pH range 5 to 7 at high carnosine to copper(II) ratios     

Conformational changes in the metal-binding sites of cardiac troponin C induced by calcium binding.

Isotope labeling of recombinant normal cardiac troponin C (cTnC3) with 15N-enriched amino acids and multidimensional NMR were used to assign the downfield-shifted amide protons of Gly residues at position 6 in Ca(2+)-binding loops II, III, and IV, as well as tightly hydrogen-bonded amides within the short antiparallel beta-sheets between pairs of Ca(2+)-binding loops. The amide protons of Gly70, Gly110, and Gly146 were found to be shifted significantly downfield from the remaining amide proton resonances in Ca(2+)-saturated cTnC3. No downfield-shifted Gly resonance was observed from the naturally inactive site I. Comparison of downfield-shifted amide protons in the Ca(2+)-saturated forms of cTnC3 and CBM-IIA, a mutant having Asp65 replaced by Ala, demonstrated that Gly70 is hydrogen bonded to the carboxylate side chain of Asp65. Thus, the hydrogen bond between Gly and Asp in positions 6 and 1, respectively, of the Ca(2+)-binding loop appears crucial for maintaining the integrity of the helix-loop-helix Ca(2+)-binding sites. In the apo- form of cTnC3, only Gly70 was found to be shifted significantly downfield with respect to the remaining amide proton resonances. Thus, even in the absence of Ca2+ at binding site II, the amide proton of Gly70 is strongly hydrogen bonded to the side-chain carboxylate of Asp65. The amide protons of Ile112 and Ile148 in the C-terminal domain and Ile36 in the N-terminal domain data-sheets exhibit chemical shifts consistent with hydrogen-bond formation between the pair of Ca(2+)-binding loops in each domain of Ca(2+)-saturated cTnC3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nitrotyrosine chelation of nuclear magnetic resonance shift probes in proteins: application to bovine pancreatic trypsin inhibitor

The interactions of Pr(III) and Eu(III) with specifically nitrated derivatives of the basic bovine pancreatic trypsin inhibitor have been studied using optical spectroscopy and nuclear magnetic resonance (NMR) at 250 and 270 MHz. Stability constants for proton and metal binding to nitrotyrosines 10 and 21 determined optically are in good agreement with those from NMR. Observations of the Eu(III)-induced NMR shifts of the ring protons of nitrotyrosine 21 allowed calibration of the magnetic interactions for this binding site. The Pr(III)-induced shifts for several resolved nonexchangeable backbone proton resonances were compared with calculated shifts using the known x-ray structure. With several simplifying assumptions, the Pr(III)-induced shifts were used to assign one alpha-CH and five NH protons to compatible sets of backbone positions which are consistent with the known pH dependence and resistance to exchange with solvent D2O. Some of the more general aspects of lanthanide-induced shifts are discussed with reference to their use in proteins. Due to the complexities of the analysis of the shift data, the most straightforward use of this technique is in conjunction with the relaxation probe Gd(III) for measurement of intramolecular distances.     

15N-labeled Escherichia coli tRNAfMet, tRNAGlu, tRNATyr, and tRNAPhe. Double resonance and two-dimensional NMR of N1-labeled pseudouridine

The N1 imino units in Escherichia coli tRNAfMet, tRNAGlu, tRNAPhe, and tRNATyr were studied by 1H-15N NMR using three different techniques to suppress signals of protons not attached to 15N. Two of the procedures, Fourier internuclear difference spectroscopy and two-dimensional forbidden echo spectroscopy permitted 1H and 15N chemical shifts to be measured simultaneously at 1H sensitivity. The tRNAs were labeled by fermentation of the uracil auxotroph S phi 187 on a minimal medium containing [1-15N]uracil. 1H and 15N resonances were detected for all of the N1 psi imino units except psi 13 at the end of the dihydrouridine stem in tRNAGlu. Chemical shifts for imino units in the tRNAs were compared with "intrinsic" values in model systems. The comparisons show that the A X psi pairs at the base of the anticodon stem in E. coli tRNAPhe and tRNATyr have psi in an anti conformation. The N1 protons of psi in other locations, including psi 32 in the anticodon loop of tRNAPhe, form internal hydrogen bonds to bridging water molecules or 2'-hydroxyl groups in nearby ribose units. These interactions permit psi to stabilize the tertiary structure of a tRNA beyond what is provided by the U it replaces.     

Strategies for the uses of lanthanide NMR shift probes in the determination of protein structure in solutio. Application to the EF calcium binding site of carp parvalbumin.

The homologous sequences observed for many calcium binding proteins such as parvalbumin, troponin C, the myosin light chains, and calmodulin has lead to the hypothesis that these proteins have homologous structures at the level of their calcium binding sites. This paper discusses the development of a nuclear magnetic resonance (NMR) technique which will enable us to test this structural hypothesis in solution. The technique involves the substitution of a paramagnetic lanthanide ion for the calcium ion which results in lanthanide induced shifts and broadening in the 1H NMR spectrum of the protein. These shifts are sensitive monitors of the precise geometrical orientation of each proton nucleus relative to the metal. The values of several parameters in the equation relating the NMR shifts to the structure are however known as priori. We have attempted to determine these parameters, the orientation and principal elements of the magnetic susceptibility tensor of the protein bound metal, by studying the lanthanide induced shifts for the protein parvalbumin whose structure has been determined by x-ray crystallographic techniques. The interaction of the lanthanide ytterbium with parvalbumin results in high resolution NMR spectra exhibiting a series of resonances with shifts spread over the range 32 to -19 ppm. The orientation and principal elements of the ytterbium magnetic susceptibility tensor have been determined using three assigned NMR resonances, the His-26 C2 and C4 protons and the amino terminal acetyl protons, and seven methyl groups; all with known geometry relative to the EF calcium binding site. The elucidation of these parameters has allowed us to compare the observed spectrum of the nuclei surrounding the EF calcium binding site of parvalbumin with that calculated from the x-ray structure. A significant number of the calculated shifts are larger than any of the observed shifts. We feel that a refinement of the x-ray based proton coordinates will be possible utilizing the geometric information contained in the lanthanide shifted NMR spectrum.     

Formate as an NMR probe of anion binding to Cu,Zn and Cu,Co bovine erythrocyte superoxide dismutases.

The binding of formate to bovine Cu,Zn superoxide dismutase has been studied by NMR spectroscopy. The distance between the copper ion and the proton covalently bound to formate has been evaluated from the broadening of the resonance of such proton. The effect on the copper-coordinated water molecule was evaluated from the bulk water relaxation effect by pulsed low-resolution NMR. The broadening of the resonance due to the formate carboxyl in the 13C NMR spectrum gave further indications about the carbon-copper distance thus providing information about the orientation of the formate ion. Changes of isotropically shifted resonances of the Cu,Co enzyme, where cobalt substitutes the native zinc, indicate that rearrangements of imidazoles of the liganding histidines occur upon binding. Transient NOE experiments gave indication of the proximity of the formate proton to resonance H of the NMR spectrum assigned to the imidazole proton of the copper-liganding His 118 of the active site. 2D NMR NOESY experiments made clear that no important rearrangement of the liganding histidines occurred in the presence of a saturating amount of formate. The absence of relevant changes of the intensity of NOE cross-peaks which are sensitive to interatomic distances in the active site revealed that only slight changes have occurred. Molecular graphics representation on the basis of all the information obtained allowed us to locate the formate in the proximity of the active site. The formate binding occurs via hydrogen bonds through the carboxylate ion and the NH groups of the side chains of Arg 141 which is external to the copper coordination sphere and faces the active site of the enzyme.     

Kinetics and mechanisms of reduction of Cu(II) and Fe(III) complexes by soybean leghemoglobin alpha

The reduction of low-molecular-weight Cu(II) and Fe(III) complexes by soybean leghemoglobin alpha was characterized using both kinetic analysis and 1H-NMR experiments. Whereas Fe(III) (CN)6(3-) was reduced through an outer sphere transfer over the exposed heme edge, all other Cu(II) and Fe(III) complexes investigated were reduced via a site-specific binding of the metal to the protein. Reduction of all metal complexes was enhanced by decreasing pH while only Fe(III)NTA reduction kinetics were altered by changes in ionic strength. Rates of reduction for both Cu(II) and Fe(III) were also affected inversely by the effective binding constant of the metal chelate used. NMR data confirmed that both Cu(II)NTA and Fe(III)NTA were bound to specific sites on the protein. Cu(II) bound preferentially to distal His-61 and Fe(III) exerted its greatest effect on two surface lysine residues with epsilon proton resonances at 3.04 and 3.12 ppm. The Fe(III)NTA complex also had a mild but noticeable line broadening effect on the distal His-61 singlet resonance near 5.3 ppm. Like hemoglobin and myoglobin, leghemoglobin might function not only as an oxygen carrier, but also as a biological reductant for low-molecular-weight Cu(II) and Fe(III) complexes.     

NMR studies on binary and ternary Pd(II) complexes formed by the growth-modulating tripeptide glycylhistidyllysine and nucleotides

The mechanism of transport of Pt(II) and Pd(II) into tissues through blood and that of their elimination in kidney is incompletely known so far. In this respect, the binding of palladium by the tripeptide glycyl-L-histidyl-L-lysine (GHL), a constituent of the human plasma, as a binary complex, and by the nucleotides 5'-IMP and 5'-GMP, as ternary complexes, has been studied by 1H and 13C NMR spectroscopy. These studies have been conducted in aqueous media and at different ligand/metal ratios. At acidic pH, resonances were observed for binary and ternary kinetically stable complexes, and binding sites in these complexes were identified by the effect of binding on chemical shifts of protons and carbon resonances. From these data, stoichiometries and structures of these complexes were proposed.     

Codon-anticodon interaction in tRNAPhe: II. A nuclear magnetic resonance study of the binding of the codon UUC

The effect of binding of the codon UUC to yeast tRNA^Phe was investigated by means of n.m.r.² spectroscopy and analytical ultracentrifugation. Binding of UUC to the transfer RNA anticodon tends to promote the aggregation of tRNA molecules; this is manifest from a line broadening in the n.m.r. experiments as well as from an increase in s_20,w the ultracentrifuge experiments. Such an aggregation of tRNA molecules was not observed upon addition of different oligonucleotides, as described in the accompanying paper. In addition to the general broadening observed in the n.m.r. spectra, specific resonances in the methyl proton spectrum as well as in the hydrogen-bonded proton spectrum are broadened or shifted upon binding of UUC.These results are explained on the basis of the premise that two different tRNA-UUC complexes can exist in solution. It is suggested that the binding of UUC tends to promote a disruption of the m⁷G₄₆ · m²₂G₂₂ base-pair and its neighbouring base-pairs.In studying the binding of U-U-U-U to yeast tRNA^Phe no resonances of protons hydrogen-bonded between the oligonucleotide and the tRNA could be detected at low temperatures. This indicates, that at these temperatures the lifetime of the tRNA-U-U-U-U complex is substantially shorter than the lifetime of the other tRNA-oligonucleotide complexes studied in this and the accompanying paper under these conditions.     

Study of the interaction between uncharged yeast tRNAPhe and elongation factor Tu from Bacillus stearothermophilis

Proton NMR studies are presented on the interaction of nonaminoacylated yeast tRNAPhe and elongation factor Tu X GTP from Bacillus stearothermophilis. From experiments in which transfer of magnetization is observed between proton spins of tRNA and the protein, it is concluded that complex formation takes place. Amino acid residues of the protein come into close contact with the base pair A5U68 and/or U52A62 of the acceptor T psi C limb of the tRNA molecule. From the line broadening of tRNA resonances, associated with complex formation, an association constant of 10(3)-10(4) M-1 is estimated. The NMR experiments do not monitor a significant conformational change of the tRNA molecule upon interaction with the protein. However, at times long after the onset of complex formation, spectral changes indicate that the upper part of the acceptor helix becomes distorted.     

Complete assignment of the imino protons of Escherichia coli valine transfer RNA: two-dimensional NMR studies in water

The imino proton spectrum of Escherichia coli valine tRNA has been studied by two-dimensional nuclear Overhauser effect spectroscopy (NOESY) in H2O solution. The small nuclear Overhauser effects from the imino proton of an internal base pair to the imino protons of each nearest neighbor can be observed as off-diagonal cross-peaks. In this way most of the sequential NOE connectivity trains for all the helices in this molecule can be determined in a single experiment. AU resonances can be distinguished from GC resonances by the AU imino NOE to the aromatic adenine C2-H, thus leading to specific base-pair assignments. In general, the NOESY spectrum alone is not capable of assigning every imino proton resonance even in well-resolved tRNA spectra. Multiple proton peaks exhibit more than two cross-peaks, resulting in ambiguous connectivities, and coupling between protons with similar chemical shifts produces cross-peaks that are incompletely resolved from the diagonal. The sequence of the particular tRNA determines the occurrence of the latter problem, which can often be solved by careful one-dimensional experiments. The complete imino proton assignments of E. coli valine tRNA are presented.     

Nuclear magnetic resonance study of hydrogen-bonded ring protons in oligonucleotide helices involving classical and nonclassical base pairs.

A study of the exchangeable ring nitrogen protons in aqueous solutions of oligonucleotide complexes involving Watson-Crick base pairs as well as Hoogsteen pairs and other nonclassical hydrogen bonding schemes shows that resolvable resonances in the low-field (-10 to -16 ppm from sodium 4,4-dimethyl-4-silapentanesulfonate) region can be detected in a variety of structures other than double stranded helices. Ring nitrogen proton resonances arising from the following hydrogen-bonding situations are reported: (1) AT and GC Watson-Crick base pairs in a self-complementary octanucleotide, dApApApGpCpTpTpT; (2) U-A-U base triples in complexes between oligo-U15 and AMP; (3) C-G-C+ base triples in complexes between oligo-C17 and GMP at acid pH; (4) s4U-A-s4U base triples in complexes between oligo-s4U15 and AMP, all of which involve both Watson-Crick and Hoogsteen base pairing to form triplexes; (5) C-C+ base pairing between protonated and unprotonated C residues in oligo-C17 at acid pH; and (6) I4 base quadruples in the four strand association among oligo-I at high salt. The behavior of the dA3G-CT3 helix is consistent with both fraying of the terminal base pairs and presence of intermediate states as the helix opens. In the monomer-oligomer complexes, under the conditions used here, the exchange appears to be governed by the dissociation rate of monomer from the complex. These findings suggest that those tertiary structure hydrogen bonds in tRNA involving ring nitrogen protons should have representative resonances in the low-field (11-16 ppm) proton NMR region in H2O.     

Carbon-13 nuclear magnetic resonance spectroscopy of high-spin iron(III) prophyrin compounds

¹³C nuclear magnetic resonance spectra have been obtained for variety of high-spin iron(III) porphyrin compounds and corresponding μ-oxo-bridged dimeric species. Large hyperfine shifts and significant line broadening are observed. The monomeric exhibit hyperfine shifts which are downfield with te exception of an upfield shift for the meso-carbon atom. Possible unpaired spin delocalization mechanisms and prospects for observing ¹³C NMR porphyrin resonances in high-spin ferrihemoproteins are discussed. Spectra reported here provide strategy for incorporation of ¹³C labels in hemoproteins either by biosynthetic or chemical means. The vinyl-CH₂ resonances of iron(III) protoporphyrin IX located 260 parts per million downfield from tetramethylsilane are especially attractive from the standpoint of chemical labeling.     

Manganese(II) as a paramagnetic probe of the tertiary structure of transfer RNA.

The effect of manganese on both the low field (10--15 ppm) and the high field (o--3 ppm) NMR spectra of unfractionated tRNA and yeast tRNAPhe has been investigated. Trace amounts of Mn2+ cause selective broadening of resonances which are assigned to specific tertiary interactions. The order in which resonances broaden is the same as the order in which they are stabilized by the addition of magnesium, namely s4U8 - A14, U33 and A58 - T54. From this we conclude that three of the strong binding sites probably are the same for both Mn2+ and Mg2+, and that these sites are located close to the tertiary interactions which are stabilized by the strongly bound metals. The broadening data, taken in conjunction with published X-ray data on yeast tRNAPhe, permit us to suggest some plausible locations for the strong binding sites.     

NMR characterization of a diamagnetic model of unliganded alpha chains from human hemoglobin.

This paper reports the reconstitution and spectroscopic characterization of a complex between alpha globin from human adult hemoglobin and protoporphyrin IX-Zn(II). Optical and proton one-dimensional (1-D) NMR spectra indicate that the prosthetic group binds in a 1:1 stoichiometry to the apoglobin in a single conformation. Using 2-D proton NMR techniques we assigned resonances corresponding to the majority of porphyrin substituents and to several side chains of amino acids in contact with the porphyrin. Analysis of nuclear Overhauser enhancement interactions between identified protons indicated that the complex contains only one rotation isomer of the prosthetic group. The diamagnetic Zn(II) ion is coordinated to the proximal histidine (His87) and does not bind O2 or CO as a sixth ligand. The ring current effects on protons from the distal valine (Val62) are considerably higher than in the liganded form providing strong evidence for a more compact ligand binding pocket relative to the carbon monoxy state. Therefore, protoporphyrin-Zn(II)/alpha globin complex is a suitable diamagnetic model for unliganded alpha chains and will be used for structure determination by NMR and modeling methods.     

1H-15N NMR studies of Escherichia coli tRNA(Phe) from hisT mutants: a structural role for pseudouridine

Escherichia coli tRNA(Phe)U39 was isolated from a specially constructed bacterial strain (DD1003/pRK3) carrying mutations in the hisT gene (the structural gene for tRNA pseudouridine synthase I) and in the pyrB gene (uracil auxotrophy). The pheU gene for tRNA(Phe) under control of the native tRNA promoter was on a multicopy plasmid and gave up to 40-fold overproduction of tRNA(Phe)U39. The double mutant permitted efficient incorporation of [3-15N]uracil, resulting in greater than 95% 15N enrichment of uracil-derived bases. 1H and 1H-15N NMR experiments were used to assign the low-field proton resonances to specific hydrogen-bonding interactions. 1H NMR assignments indicate that tRNA(Phe)U39 has a structure similar to that of native tRNA(Phe) except in the anticodon region where replacement of pseudouridine (psi) at position 39 with uridine (U) destabilizes hydrogen-bonding interactions at the base of the anticodon stem. We propose that U----psi modifications further stabilize interactions normally available to U by providing an additional locus for hydrogen bonding to the pyrimidine ring.     

Axial histidyl imidazole non-exchangeable proton resonances as indicators of imidazole hydrogen bonding in ferric cyanide complexes of heme peroxidases

Proton NMR spectra of a model of low-spin cyanide complexes of ferric hemoproteins indicate that two broad single-protein resonances from the axial imidazole can be resolved outside the diamagnetic spectral region. Upon deprotonation of the imidazole in the model, the upfield resonance shifts dramatically to higher field, suggesting that its position may reflect the degree of hydrogen bonding or proton donation of the imidazole. Met-cyano myoglobin reveals a pair of such broad peaks in the regions expected for an essentially neutral axial imidazole. In the cyano complexes of horseradish peroxidase and cytochrome c peroxidase, a pair of single-proton resonances are located which are assigned to the same imidazole protons on the basis of their linewidth and shift changes upon altering the heme substituents. The upfiled proton, however, is found at much higher field than in metMbCN. The upfield bias of this resonance is taken as evidence for appreciable imidazolate character for the axial ligand in these heme peroxidases.