Ring NH Protons Research Articles

A ProtonT1,T2, and NOE Study of Relative Motion of the Indole Ring of Tryptophans in Gramicidin Analogs Incorporated into SDS Micelles

The proton spin–lattice relaxation time, spin–spin relaxation time, and NOE of the indole ring NH proton of tryptophan residues have been determined for seven analogs of gramicidin A incorporated into SDS micelles. The data obtained indicate that the motion of the indole rings systematically decreases, proceeding from the aqueous interface to the interior of the micelle.

H NMR probes for inter-segmental hydrogen bonds in myoglobins.

NMR signals arising from the HisB5 N delta H and HisEF5 N epsilon H protons in sperm whale skeletal and horse heart myoglobins have been located for the first time in the downfield shifted portion of the spectra. The shifts and hydrogen exchange rates indicate that these His imidazole ring NH protons are involved in the inter-segmental hydrogen bonds of the protein in solution, as demonstrated by a crystallographic study [Takano, T. (1977) J. Mol. Biol. 220, 381-399]. The assigned His imidazole ring NH proton resonances can serve as new sensitive structural probes in the study of the local conformation of myoglobin. The applicability of the NMR spectral parameters in the study of the tertiary structure of apomyoglobin, the denaturation of the protein, and the protein stability of sperm whale and horse myoglobins is presented in some detail.

Evidence of Histidine Coordination to the Catalytic Ferrous Ion in the Ring-Cleavlng 2,2′,3-Trihydroxybiphenyl Dioxygenase from the Dibenzofuran-Degrading Bacterium Sphingomonas sp Strain RW1

The 1H NMR spectra of an aromatic ring-cleaving extradiol dioxygenase, 2,2′,3-trihydroxybiphenyl dioxygenase of the dibenzofuran-degrading bacterium Sphingomonas sp. strain RW1, are reported. In the catalytically active reduced form of the monomeric enzyme (MW=32 kDa), three broad strongly downfield shifted signals were observed, two of which disappeared in D 2O solution. Their shifts and linewidths are consistent with ring NH and meta-like protons of coordinated histidines. These signals show strong sensitivity to the presence of the substrate. The oxidized form of the enzyme shows no hyperfine shifted signals. It is suggested that the high spin Fe(II) ion present in the active form of the enzyme is coordinated by at least two histidines. This is the first report of hyperfine shifted NMR signals being detected for an extradiol dioxygenase.

Long-acting dihydropyridine calcium antagonists. Part 8. A comparison of the pharmacological and pharmacokinetic properties of amlodipine with its carba and thio-bioisosteres

In order to evaluate the contribution to the overall pharmacokinetic and pharmacological profile of amlodipine made by the side-chain ether oxygen atom and the intramolecular hydrogen bond to the DHP ring NH proton, the profile of amlodipine was compared with that of its carba and thio bioisosteres. Replacing the side-chain oxygen by carbon dramatically reduces in vitro calcium antagonist potency, an effect which may be attributed to the loss of a through-bond inductive effect on the DHP ring NH proton, while both the thio and carba analogues show lower in vitro selectivity than amlodipine for vascular over cardiac tissue. On intravenous administration to anaesthetised dogs, compounds 2 and 3 both exhibit marked depression of myocardial contractility at doses equal or close to their ED50 for reduction of coronary vascular resistance. The plasma clearances of amlodipine and analogues 3 and 4 are similar, suggesting that the conformation adopted by the 2-sidechain has little influence on this parameter although bulk and polarity are important. However, compounds 3 and 4 have markedly lower volumes of distribution (6 and 8 dm3 kg–1, respectively) than amlodipine (25 dm3 kg–1) and consequently shorter half-lives; this may be a consequence of their inability to form an intramolecular hydrogen bond.

Nuclear magnetic resonance studies on structure and breathing dynamics of transfer RNA.

The hydrogen-bonded ring NH protons of the complementary base pairs in Escherichia coli isoleucine tRNA have been studied by 500 MHz nuclear magnetic resonance. These protons generate a set of about 30 well-resolved resonances in the -10p.p.m. to-15 p.p.m. region of the spectrum and can be observed in H2O solutions by Fourier transform NMR using the Redfield 214 observation pulse. Each of these low field protons has been assigned to its respective base pair in the tRNA by sequential nuclear Overhauser effect connectivity to its nearest neighbours. The helix-coil opening rates for several individual base pairs have been determined by monitoring the solvent-exchange contribution to the inversion recovery rate or the saturation recovery rate of the assigned ring NH peaks. Contrary to current theory, the two most rapidly opening base pairs in the molecule are located not at the helix termini but in the interior of helices.

Direct assignment of the dihydrouridine-helix imino proton resonances in transfer ribonucleic acid nuclear magnetic resonance spectra by means of the nuclear Overhauser effect.

The NMR resonances from the hydrogen-bonded ring NH protons in the dihydrouridine stem of Escherichia colt tRNA1Val have been assigned by experiments involving the nuclear Overhauser effect (NOE) between adjacent base pairs. Irradiation of the 8-14 tertiary resonance produced a NOE to base pair 13. Irradiation of the CG13 ring NH produced NOEs to base pairs 12 and 14. Similarly, base pair 12 was shown to be dipolar coupled to 11 and 13, and base pair 11 was found to be coupled to 10 and 12. These sequential connectivities led to the assignment of CG13 at -13.05 ppm, UA12 at -13.84 ppm, CG11 at -12.23 ppm, and GC10 at -12.60 ppm. The results are compared with previous, less direct assignments for these four base pairs and with the expected proton positions from the crystal structure coordinates for this helix.

Nuclear Overhauser effect study and assignment of D stem and reverse-Hoogsteen base pair proton resonances in yeast tRNAAsp.

Nuclear Overhauser effects (NOEs) in yeast tRNAAsp were found for all four GU and G psi base pairs. NOEs of both reverse-Hoogsteen pairs were identified by comparison with a purine C8 deuterated sample. Several NOEs involving these resonances were also found which are clearly between single protons on adjacent base pairs. These interbase NOEs, combined with the assumption of reasonable similarity between the structure of yeast tRNAAsp and that of yeast tRNAPhe, lead to unambiguous assignment of many resonances including all the ring NH and C2 protons in the D stem. The stability of the stem at 28 degrees C, as recently deduced by Moras et al (Nature 288 669-674), from x-ray diffraction is confirmed. Assignments of the ring NH resonances of T54-A58 and of a G psi pair are made for the first time.

Identification of tertiary base pair resonances in the nuclear magnetic resonance spectra of transfer ribonucleic acid

The low-field hydrogen-bond ring NH proton nuclear magnetic resonance (NMR) spectra of several transfer ribonucleic acids (tRNAs) related to yeast tRNAPhe have been examined in detail. Several resonances are sensitive to magnesium ion and temperature, suggesting that they are derived from tertiary base pairs. These same resonances cannot be attributed to cloverleaf base pairs as shown by experimental assignment and ring current shift calculation of the secondary base pair resonances. The crystal structure of yeast tRNAPhe reveals at least six tertiary base pairs involving ring NH hydrogen bonds, which we conclude are responsible for the extra resonances observed in the low-field NMR spectrum. In several tRNAs with the same tertiary folding potential and dihydrouridine helix sequence as yeast tRNAPhe, the extra resonances from tertiary base pairs are observed at the same position in the spectrum.

Helix-Coil Transition of the Self-Complementary dG-dG-dA-dA-dT-dT-dC-dC Duplex

The helix-coil transition of the octanucleotide self-complementary duplex dG-dG-dA-dA-dT-dT-dC-dC has been monitored at the Watson-Crick protons, the base and sugar nonexchangeable protons and the backbone phosphates by high-resolution nuclear magnetic resonance (NMR) spectroscopy. The melting transition of the octanucleotide monitored by ultraviolet absorbance spectroscopy is characterized by the thermodynamic parameters delta H degree = -216.7 kJ/mol and delta S degree (25 degrees C) = -0.632 KJ mol-1 K-1 in 0.1 M NaCl, 10 mM phosphate solution. Correlation of the transition midpoint values monitored by the ultraviolet absorbance studies at strand concentrations below 0.2 mM and by NMR studies at 5.3 mM suggest that both methods are monitoring the octanucleotide duplex-to-strand transition. The NMR spectra of the Watson-Crick ring NH protons of the octanucleotide duplex have been followed as a function of temperature. The resonance from the terminal dG.dC base pairs broadens out at room temperature while the resonances from the other base pairs broaden simultaneously with the onset of the melting transition. The nonexchangeable base and sugar H-1' protons are resolved in the duplex and strand states and shift as average peaks through the melting transition. The experimental shifts on duplex formation have been compared with calculated values based on ring-current and atomic diamagnetic anisotropy contributions for a B-DNA base-pair-overlap geometry in solution. Several nonexchangeable proton resonances broaden in the fast-exchange region during the duplex-to-strand transition and the excess widths yield a duplex dissociation rate constant for the octanucleotide of 1.9 x 10(3) s-1 at 32 degrees C (fraction of duplex = 0.86) in 0.1 M NaCl, 10 mM phosphate buffer. The 31P resonances of the seven internucleotide phosphates are distributed over 0.6 ppm in the duplex state, shift downfield during the duplex-to-strand transition and undergo additional downfield shifts during the stacked-to-unstacked strand transition with increasing temperature.

Proton magnetic resonance studies of ribonuclease T 1. Assignment of histidine-40 peak and analysis of the active site

Nuclear magnetic resonances of the C-2 protons of the three histidine residues in ribonuclease T 1 have been studied at 360 MHz as a function of pH to discuss the structure of the active site. Comparison of the order of deuterium exchange of the histidine peaks with tritium incorporation rates into individual histidines of the enzyme leads to the unambigous assignment of one of the C-2 proton peaks to histidne-40. It has been concluded that histidine-40 is in the active site, interacting with a charged group of pK 4.1, which is replaced by the phosphate group of guanosine-3′-monophosphate in the enzyme-inhibitor complex. Histidine-92 is most likely a binding site for the complex, where the existence of a hydrogen bond between N-7 of the inhibitor and the ring NH proton of the histidine is suggested on the basis of NMR data.

NMR investigation of proton exchange in transfer rna by high resolution NMR

Summary High resolution (300 MHz) nmr has been used to follow the exchange-out of ring NH protons involved in watson-Crick secondary structure base pairs in yeast tRNA3Leu and tRNAPhe. At 4°C the ∼20 resonances observed in the low field region of the spectrum (11 to 15 ppm) exchange with the solvent (D2O) in less than 300 sec. Because the exchange—out is so rapid, we are unable to follow the behavior of imino protons in specific watson-Crick base pairs. The 20–40 very slowly (1 hr.) exchanging protons observed by tritium exchange must be assigned to amino protons.

Identification of a unique ethidium bromide binding site on yeast tRNAPhe by high resolution (300 MHz) nuclear magnetic resonance.

The binding of ethidium bromide to yeast tRNAPhe has been studied by high resolution (300 MHz) proton nuclear magnetic resonance. Under appropriate experimental conditions one ethidium bromide is bound to each tRNA and two resonances from ring NH protons are shifted upfield. These observations taken in conjunction with the low-field spectrum of yeast tRNAPhe show that the unique ethidium bromide binding site is located between base pairs AU6 and AU7 of the amino acid acceptor stem. This information should be of value in understanding the way in which ethidium bromide binding alters the biochemical properties of the tRNA molecules.

Open states in native polynucleotides: II. Hydrogen-exchange study of cytosine-containing double helices

Hydrogen-exchange studies of I · C and G · C double helices were carried out to test the generality of conclusions reached previously in studies of adenine-containing polymers (preceding paper). The cytosine amino group shows hydrogen-exchange behavior similar to the analogous group in adenine; a pH-independent pathway and a parallel general catalysis pathway require prior separation of the base-pair and pre-equilibrium protonation at the ring N. The cytosine amino group does, however, display greater sensitivity to specific and to general catalysis than found for adenine. In the G · C helix, the ring NH proton of guanine exchanges at the opening-limited rate, as does the analogous proton in A · U and A · T pairs, while the guanine amino protons exchange without a prior opening of structure. From the observed exchange rates and the known chemistry for the pH-independent reaction, one can calculate equilibrium opening constants of 4 × 10 −3 for poly(rI) · poly(rC) and perhaps one tenth of that for poly(rG) · poly(rC). Also the opening rate constant for the G · C helix is 0.01 s −1. These results, when applied to published exchange curves for DNA, indicate an equilibrium opening constant of 0.005, an opening rate constant of 0.04 s −1, and a closing rate constant of 10 s −1. (All values refer to studies at 0 °C.) These values point to the same kind of traveling-loop model for base-pair opening discussed previously for the opening reactions in adenine-containing double helices.

NMR evidence for tertiary structure base pair in E. coli tRNA involving S4U8.

IN previous nuclear magnetic resonance (NMR) studies of tRNA we observed anomalously low field resonances (14.5–15.0 p.p.m. downfield from the usual standard 2,2-dimethylsilapentane-5-sulphonate (DSS)) in the spectra of many, but not all, Escherichia coli tRNA1–3 but yeast tRNA we examined never exhibited such resonances1,2. The fact that approximately 60% of the E.coli tRNA are class I and have a 4-thiouridine (S4U) residue at position 8 (refs 4 and 5), whereas yeast tRNA do not, raised the possibility that the anomalous low field resonances in E. coli tRNA might be due to ring NH protons of S4U8 in a tertiary structure base pair.

The molecular mechanism of thermal unfolding of Escherichia coli formylmethionine transfer RNA

The molecular mechanism of thermal unfolding of Escherichia coli tRNA fMet (in 0.17 m-NaCl without Mg 2+) has been elucidated by a combination of relaxation kinetics and proton nuclear magnetic resonance spectroscopy. We measured the n.m.r. ‡ ‡ n.m.r., nuclear magnetic resonance; hUra, dihydrouracil. spectrum of the hydrogen-bonded ring NH protons at different temperatures and found that the resonances assigned to each arm of the cloverleaf broaden and disappear together, yielding four distinct n.m.r. “melting” transitions. Temperature-jump measurements in the same solvent showed five co-operative melting transitions, varying in relaxation time from a few microseconds to ten milliseconds. The relaxation and n.m.r. measurements were correlated by the following model. When the lifetime of a hydrogen-bonded proton in a helix is five milliseconds, its n.m.r. line will be broadened to approximately twice its intrinsic low-temperature width and appear to “melt”. The helix dissociation time constants of the relaxation effects were extrapolated by the Arrhenius equation to lower temperatures where their values were five milliseconds. The correlation of extrapolated dissociation time constants with n.m.r. melting of specific helices allowed assignments of the structural basis for each relaxation effect. The results show that the principal path for the reversible thermal unfolding of tRNA 1 fMet under these solution conditions is first, transient opening of the dihydrouridine helix, followed by simultaneous melting of the dihydrouridine helix and a “tertiary” interaction, which does not correspond to a cloverleaf helix. The tertiary interaction is much less stable in tRNA 3 fMet, with T m lowered by 16 °C from tRNA 1 fMet. The sequence of melting steps at higher temperatures is the same in the two isoacceptors: first the TΨC helix melts, followed by the anticodon helix and finally the acceptor stem helix. Thermodynamic and kinetic parameters are reported for these steps. The method of sequential melting, combining n.m.r. and relaxation kinetic techniques, is a powerful procedure for elucidating RNA secondary structure. In addition, this method allows assignment of many hydrogen-bonded ring NH proton resonances that are unresolved in the low-temperature spectrum.

High resolution proton NMR study of an isolated fragment of R17 bacteriophage mRNA.

HIGH resolution nuclear magnetic resonance (NMR) spectra of the hydrogen-bonded protons in helical regions of nucleic acids are an informative monitor of the base pairing1. Studies of about 10 purified tRNAs and of model systems have shown that the ring NH protons of uracil and guanine, when involved in Watson-Crick base pairing, give resonances between −15 and −11 p.p.m. downfield from DSS1,2. Since there is only one such proton in each base pair, the intensity in this spectral region is a direct measure of the number of base pairs. Simple rules have been derived to predict the NMR spectra originating from these protons on the basis of ring currents from the adjacent bases and for tRNA the comparison between predicted and observed spectra has strongly supported the clover leaf model of base pairing. In addition, ring current shifts from bases adjacent to the helical region provided information about the structure at the end of the helix. Here we apply this approach to the secondary structure of the R17 fragment discussed in the preceding paper3. We prepared 0.5 mg of the R17 fragment according to a somewhat modified method of Bernardi and Spahr4, and measured the NMR spectrum of a 25 µl sample.

Conformation of charged and uncharged tRNA.

The effect of aminoacylation on the conformation of yeast tRNA(Phe) was investigated by high-resolution (300 MHz) proton nuclear magnetic resonance (NMR) spectroscopy. Resonances in the low-field (-11 to -15 ppm) region of the spectra are due to ring NH protons of Watson-Crick base pairs, and to a very high degree of approximation (within 0.05 ppm) the low-field spectra of tRNA(Phe) and phenylalanyl-tRNA(Phe) are identical. From this observation and analysis of the low-field NMR spectra we conclude that the secondary structures of the two tRNAs are identical with respect to base-pairing schemes and interbase distances in the helical region (0.1-0.2 A). Several tertiary structural features, including conformation of the dihydro-U loop, conformation of the minor loop, relative orientations of the acceptor and the TPsiC stems, dihydro-U and anticodon stems, and probably conformation of the anticodon loop are shown to be the same in tRNA(Phe) and phenylalanyl-tRNA(Phe). Our results leave little remaining opportunity for changes in tertiary structure that would not have been observed by the NMR method.

Determination of secondary and tertiary structural features of transfer RNA molecules in solution by nuclear magnetic resonance.

High-resolution 300-MHz proton nuclear magnetic resonance spectra of the hydrogen-bounded protons in three different purified tRNA molecules are presented. The resonances in the region between -11 and -15 ppm from 2,2-dimethyl-2-silapentane-5-sulfonate (DSS) are assigned to the ring NH protons of specific base pairs by two approaches. First, intrinsic positions of -14.8 ppm and -13.7 ppm are taken for the AU and GC ring NH protons, respectively, and the spectra are calculated by including ring current shifts from the nearest neighbors. The spectra calculated in this way on the basis of the cloverleaf are in good agreement with the observed. Second, fragments of yeast tRNA(Phe) were obtained, which helped in assignments of the spectrum of intact molecules. The close agreement strongly supports the cloverleaf model. Tertiary structural features were determined in a few cases where the ring currents at the terminal base pairs of helical regions depended upon stacking of the helices. In this way, we were able to show that in Escherichia coli tRNA(Glu) the CCA stem forms a continuous helix with the TPsiC stem, which is in accord with the preliminary x-ray structure of yeast tRNA(Phe), suggesting that this stacking is observed in solution and may be a general property of different tRNA molecules. Similar reasoning suggests that in E. coli tRNA(fMet) G-27 is stacked upon the dihydrouridine helix.

Assignment of the low field proton nuclear magnetic resonance spectrum of yeast phenylalanine transfer RNA to specific base pairs

High-resolution proton nuclear magnetic resonance spectra at 220 and 300 MHz have been used to investigate the base-pairing structure of fragments of yeast tRNA Phe, of chemically modified tRNA Phe and of intact tRNA Phe. To a very good approximation the positions of the fragment spectra are additive within 0·2 part per million, indicating that factors responsible for certain structural features in the intact molecule are already present in the smaller fragments (half molecules, hairpins and 3 4 molecules). A simple first-order ring-current shift theory taken in conjunction with the cloverleaf model for tRNA Phe (RajBhandary et al., 1967) has been used to predict the low-field (− 15 to −11 part per million) nuclear magnetic resonance spectra and make assignments of the resolved resonances to ring NH protons of specific base pairs. The general agreement between the predicted and observed spectra to within 0·2 part per million confirms in detail the cloverleaf model for the secondary structure of tRNA Phe in solution. It is also established that ring-current shifts are the principal factor responsible for the wide range of shifts observed in the low-field spectra. As a result it is evident that the resonances are very sensitive to small changes in the secondary structure and in some cases changes in the interbase distance as small as 0·2 Å could easily be detected. It is also clear from the analysis that certain of the resonances are sensitive to the tertiary structure of the molecule and specific examples are discussed. As with our previous study, we find no evidence for any strong Watson-Crick type base pairs beyond those predicted by the cloverleaf structure.