Ring-current shifts in the 300 MHz nuclear magnetic resonance spectra of six purified transfer RNA molecules

We present the 300 MHz high-resolution proton nuclear magnetic resonance spectra of the ring NH hydrogen-bonded protons of six purified tRNAs. Good agreement was obtained between the observed spectra and those computed on the assumption of the suitable cloverleaf models. In the computation it is assumed that the hydrogen-bonded ring NH in each type of base pair has an intrinsic position with respect to 2,2-dimethyl-2-silapentane-5-sulfonate, i.e. in A·U it is at ?14·8 parts per million, in G·C at ?13·7 parts per million and in A·Ψat ?13·5 parts per million. The shifts of these resonances from these positions are calculated by including ring current fields from the nearest neighbors. The agreement is very good, adding support to our earlier findings that there is no evidence for additional Watson-Crick base pairs detected beyond those in the cloverleaf. In general, resolved resonances are fitted by the computed spectra to within ±0·2 part per million showing that there is no need for any additional physical mechanism to explain the nuclear magnetic resonance positions. Hence, the nuclear magnetic resonance spectra can be interpreted in terms of the structure of their neighbors and in a few important cases this has been particularly valuable in understanding the structure beyond the end of a helical region. In the tRNA^Glu_E.coli′ for example, the positions of the resonances in A·U no. 7 and A·U no. 49 at the interior ends of the acceptor and -T-Ψ-C- stems, respectively, strongly suggest that these two stems are in a continuous helix. Other structural effects at the ends of the helical regions are also suggested by the nuclear magnetic resonance spectra.     

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 $\text{3}\text{4}$ 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.     

Tritium exchange studies of transfer RNA in native and denaturated conformations

Tritium exchange was used as a probe of transfer RNA structure in experiments with unfractionated tRNA (tRNA^Unfrac and homogeneous tRNA₃^Leu from bakers' yeast. Exchange kinetics were measured over a range of ionic conditions that vary in ability to stabilize the secondary and tertiary structure of tRNA. The native conformations of both samples show the same kinetics of exchange. The kinetics for tRNA₃^Leu trapped in a denatured state in a “native” solvent are much faster, reflecting the conformation and not the ionic medium. In 0.1 M-Na⁺, where tRNA₃^Leu is denatured, the kinetics for tRNA^Unfrac are intermediate between those for native and denatured tRNA₃^Leu, suggesting that in this solvent at 0 °C some tRNAs are denatured whereas other are still native. Upon further lowering of Na⁺ concentration, tRNA^Unfrac shows increasingly faster exchange, suggesting complete electrostatic denaturation of the tertiary structure of all the tRNAs in the sample, and even disruption of secondary structure.Extrapolation of the essentially linear early-time kinetics to zero time provides minimal estimates of the number of slowly exchanging hydrogens. For native tRNA₃^Leu the number is 111±2 hydrogens, whereas for the trapped denatured conformation it is only 95±2. This difference reflects a smaller number of hydrogen-bonded bases in the denatured conformation. In 1 M-Na⁺, 101±2 slowly exchanging hydrogens are found for the native tRNA₃^Leu conformation, suggesting an incompletely formed native structure. For native tRNA^Unfrac the comparable number is 101±3. These numbers of slowly exchanging hydrogens in the native conformations are consistent with tertiary structural hydrogen-bonding. Furthermore, this tertiary structure must be responsible for the slower exchange by native tRNA. The observed numbers of exchangeable hydrogens provide a basis for comparison of hydrogen-bonding interactions in native and denatured tRNA conformations.The mechanism of renaturation was also investigated, using tritium exchange as a monitor of perturbation of base pairing during the transition. When tRNA^Unfrac in low Na⁺ is renatured by addition of Mg²⁺ during tritium exchangeout, a burst of exchange or “spillage” of tritium is detected. This suggests that a fraction of the base pairs of the rapidly renaturing tRNAs in the mixture is disrupted during renaturation. In that event, and by analogy with tRNA₃^Leu, part of the base-pairing arrangement of the denatured conformations may not be preserved in the native state; and if the native conformation includes the full “cloverleaf” pattern of secondary structure, that pattern may not be intact in some denatured conformations.     

Evaluation of base-pairing schemes for E. coli 5S RNA by 400 MHz proton nuclear magnetic resonance spectroscopy

The number of base pairs in the denatured “B” form of $\text{E. coli}$ 5S RNA has been determined directly from 400 MHz high resolution proton nuclear magnetic resonance spectroscopy. The experimental NMR spectrum from ?11.6 to ?14.5 ppm from a sodium 2,2-dimethyl-2-silapentane sulfonate reference can be simulated by a theoretical spectrum consisting of 33 Lorentzian lines of equal width (corresponding to 33 base pairs) at 26°C. This result is inconsistent with previously proposed secondary structures of 17 and 23 base pairs, but is readily adapted to the Luoma-Marshall cloverleaf secondary structure.     

Kinetics of the renaturation of yeast tRNA3 leu.

Yeast tRNA₃^Leu is one of several tRNA molecules which can adopt a stable, biologically inactive, denatured conformation. The circular dichroism of the native and denatured conformers differs, providing the basis for the present study of the mechanism for the renaturation process. Conversion of the denatured structure to the native takes place in two steps: a rapid change occurring immediately on addition of Mg⁺⁺, followed by a slower, strongly temperature-dependent step which returns the molecule to its biologically active state. Optimal kinetic data for the second step could be obtained at 285 nm. Analysis of the time dependence of Δε₂₈₅ by the Guggenheim method demonstrated that this step follows first-order kinetics. The temperature dependence of the rate constants over the range 32–41°C yielded the following parameters for the rate-limiting step: E_a = 69 kcal/mole, ΔH^? = 69 kcal/mole, and ΔS^? = 146 cal/mole deg. Values of this magnitude are typical of order—order transitions in nucleic acids.     

High resolution nuclear magnetic resonance studies of the active site of chymotrypsin. I. The hydrogen bonded protons of the "charge relay" system

High resolution proton nuclear magnetic resonance has been used to observe protons at the active site of chymotrypsin A_δ and at the same region of chymotrypsinogen A. A single resonance with the intensity of one proton is located in the low field region of the nuclear magnetic resonance spectrum. This resonance is observed in H₂O solutions but not in ²H₂O. On going from low to high pH the resonance titrates upfield 3 parts per million in both proteins and has a pK of 7.5. The titration can be prevented by alkylating His57 with either of two active site directed chloromethyl ketones. Using these data the proton resonance has been assigned to a proton in a hydrogen bond between His57 and Asp102. Further confirmation of this assignment lies in the observation of a similar resonance in this same low field region of the nuclear magnetic resonance spectrum of trypsin, trypsinogen, subtilisin BPN′ and α-lytic protease all of which have the Asp-His-Ser triad at their active sites.This proton resonance in chymotrypsin A_δ was used as a probe to monitor the charge state of the active site upon formation of a stable acyl-enzyme analogue N²(N-acetylalanyl)-N¹benzoylcarbazoyl-chymotrypsin A_δ. In this derivative the His-Asp proton resonance titrates from the same low pH end point as in the native enzyme, ?18 parts per million, to a new high pH end point of ?14.4 parts per million (versus ?15.0 parts per million in the native enzyme). The difference of 0.6 parts per million in the high pH end points between the native and acyl enzyme is interpreted as supporting the suggestion that a hydrogen bond exists between Ser195 and His57 in the native enzyme and zymogen.We conclude from these studies that the charge relay system from Asp102 across His57 to Ser195 is intact in chymotrypsin A_δ and chymotrypsinogen A, and that, in the native enzyme, it slightly polarizes Ser195.     

Nuclear magnetic resonance studies on oligo-deoxyribonucleotides containing thedam methylation site GATC. Adenine methylation of d(GGATCC) does not change the helix geometry

We have studied the conformation of two hexanucleotides d(GGATCC) and d(GGm⁶ATCC) using proton nuclear magnetic resonance. Nuclear Overhauser effect measurements show that d(GGATCC) assumes a normal right handed B helix. The single and double strand resonances are in fast exchange on a proton nuclear magnetic resonance time scale. For d(GGm⁶ATCC), up to the Tm separate resonances are observed for each state, indicating slow exchange, though above the Tm it becomes more rapid. The orientation of the adenosine methyl-amino group, preferentiallycis to N¹, hinders base pair formation.The connectivities of the resonances of the two states were established by saturation transfer experiments. At 0°C irradiation of the m⁶ A-T imino proton gives an nuclear Overhauser effect to AH² showing that base pairing is Watson-Crick. Intra and interresidue nuclear Overhauser effects starting from the 3′ terminus show that the helix is right handed and in the B-form.The results on the two oligomers demonstrate that adenosine methylation induces little or no change in the conformation of the helix, but reduces the Tm from 45° to 32°C and slows the opening and closing of the m₆A.T base pair by a factor of about 100.     

NMR Studies of the Interaction of Bleomycin with (dC-dG)3

Abstract

The interaction of bleomycin A₂ and Zn(II)-bleomycin A₂ with the oligonucleotide (dC-dG)₃ has been monitored by nuclear magnetic resonance spectroscopy. Binding of the drug to the oligonucleotide is indicated by an upfield shift of the bithiazole proton resonances consistent with partial intercalation of this group between base pairs. The effect of temperature and ionic strength on the binding of both free bleomycin and the Zn(II) complex has been studied. Consistent with earlier studies on polynucleotides, the rate of exchange between the free drug and the drug-oligonucleotide complex is rapid on the ¹H NMR chemical shift time scale. Binding of the oligonucleotide induced changes in resonances assigned to protons in the metal-binding region of Zn(II)-bleomycin. Intermolecular nuclear Overhauser effect enhancements between bleomycin and the oligonucleotide have not been detected.     

The thermal unfolding of ribonuclease A A 13C NMR study

The ¹³C nuclear magnetic resonance (NMR) spectra of ribonuclease A over the pH range 1–7 and between 6 and 70°C reveal many of the details of its reversible unfolding. Although the unfolding may loosely be described as ‘two-state’, evidence is presented for intermediate unfolding stages at least 10°C on either side of the main unfolding transition, particularly at low pH. The first residues to unfold are 17–24, in agreement with other results. The C-terminal region shows a steeper temperature dependence of its unfolding than does the main transition, which itself is shown to lead at all pH values to a semi-structured but internally flexible state which is far from being truly random-coil. This is confirmed by measurements of T₁ and of nuclear Overhauser enhancement. Indeed, even at pH 1.1 and 70°C there is evidence for considerable motional restriction of cysteine and proline residues, amongst others.The native protein has more variability of structure at low pH than at neutral pH, and also interchanges more rapidly with the semi-structured, denatured state.     

Resonance assignment and structural analysis of acid denatured E. coli [U-15N]-glutaredoxin 3

Virtually complete sequence specific ¹H and ¹⁵N resonance assignments are presented for acid denatured reduced E. coli glutaredoxin 3. The sequential resonance assignments of the backbone rely on the combined use of 3D F₁-decoupled ROESY-¹⁵N-HSQC and 3D ¹⁵N-HSQC-(TOCSY-NOESY)-¹⁵N-HSQC using a single uniformly ¹⁵N labelled protein sample. The sidechain resonances were assigned from a 3D TOCSY-¹⁵N-HSQC and a homonouclear TOCSY spectrum. The presented assignment strategy works in the absence of chemical exchange peaks with signals from the native conformation and without ¹³C/¹⁵N double labelling. Chemical shifts, ³J(H, NH) coupling constants and NOEs indicate extensive conformational averaging of both backbone and side chains in agreement with a random coil conformation. The only secondary structure element persisting at pH 3.5 appears to be a short helical segment comprising residues 37 to 40.Abbreviations HSQC heteronuclear single quantum coherence - NMR nuclear magnetic resonance - NOE nuclear Overhauser effect - NOESY two-dimensional NOE spectroscopy - ROE nuclear Overhauser effect in the rotating frame - ROESY two-dimensional ROE spectroscopy - TOCSY total correlation spectroscopy - TPPI time proportional phase incrementation Correspondence to: G. Otting     

Oligonucleotide binding to the native and denatured conformers of yeast transfer RNA-3 Lea

The equilibrium binding patterns of complementary oligonucleotides to the native and denatured conformers of yeast transfer RNA₃^Leu have been determined. The pattern of binding to the native conformer follows that observed previously with other tRNAs. The results indicate that the anticodon loop and 3′ terminus are free in solution, and that all stems of the cloverleaf appear intact, although the dihydrouracil and “extra arm” stems are sufficiently weak to be subject to competitive binding by the probe oligomers. The T ΨC loop is also inaccessible to oligomer binding, while the dihydrouracil loop shows a low level of binding suggestive of oligomer competition with existing RNA structure. By contrast, in the denatured conformer the dihydrouracil loop and stem show strong oligomer binding characteristics of random RNA segments, whereas the anticodon loop no longer binds complementary oligomers. Binding to other regions remains unchanged, suggesting that the three major cloverleaf stems are intact. These observations are used as a basis for consideration of models for the two conformers.     

Physical properties of cytoplasmic membrane-associated DNA

Some of the physical properties of a cytoplasmic membrane-associated DNA isolated from a diploid human lymphocyte cell line have been examined. Cytoplasmic membrane-associated DNA extracted from lymphocytes labeled with either [³H]or [¹⁴C]thymidine had a specific activity lower than nuclear DNA extracted from the same cells. Analysis of cytoplasmic membrane-associated DNA in the electron microscope shows that the molecules are linear and have a mean length of 1·75 μm; the average sedimentation coefficient of this DNA is 16·6 S, which corresponds to a molecular weight of 4·2×10⁶. Cytoplasmic membrane-associated and nuclear DNA band at identical positions in both neutral and alkaline CsCl gradients with buoyant densities of 1·699 g/ml and 1·752 g/ml, respectively. Native cytoplasmic membrane-associated DNA is double-stranded and has a mole fraction of guanine plus cytosine of 40± l %. Sheared, denatured cytoplasmic membrane-associated DNA reassociates as two distinct fractions whose rates of reassociation differ by about four decades: the complexity of the reassociation of this DNA tends to rule out the possibility that it arises from either mycoplasmal or viral contamination of our cell cultures. The slowly reassociating fraction of cytoplasmic membrane-associated DNA reassociates about ten times faster than the unique sequences of nuclear DNA. This could represent potential genetic information for about 100,000 diverse genes of 1000 nucleotide pairs each. At present the function of cytoplasmic membrane-associated DNA in these cells is unknown.     

Sequence and conformational effects on imino proton exchange in A.T- and A.U-containing DNA and RNA duplexes

¹H-nmr relaxation has been used to study the effect of sequence and conformation on imino proton exchange in adenine–thymine (A · T) and adenine–uracil (A · U) containing DNA and RNA duplexes. At low temperature, relaxation is caused by dipolar interactions between the imino and the adenine amino and AH2 protons, and at higher temperature, by exchange with the solvent protons. Although room temperature exchange rates vary between 3 and 12s^?1, the exchange activation energies (E_α) are insensitive to changes in the duplex sequence (alternating vs homopolymer duplexes), the conformation (B-form DNA vs A-form RNA), and the identity of the pyrimidine base (thymine vs uracil). The average value of the activation energy for the five duplexes studied, poly[d(A-T)], poly[d(A) · d(T)], poly[d(A-U)], Poly[d(A) · d(U)], and poly[r(A) · r(U)], was 16.8 ± 1.3 kcal/mol. In addition, we find that the average E_α for the A.T base pairs in a 43-base-pair restriction fragment is 16.4 ± 1.0 kcal/mol. This result is to be contrasted with the observation that the E_α of cytosine-containing duplexes depends on the sequence, conformation, and substituent groups on the purine and pyrimidine bases. Taken together, the data indicate that there is a common low-energy pathway for the escape of the thymine (uracil) imino protons from the double helix. The absolute values of the exchange rates in the simple sequence polymers are typically 3–10 times faster than in DNAs containing both A · T and G · C base pairs.     

Quantitative determination of the conformation of cyclic 3',5'-adenosine monophosphate in solution using lanthanide ions as nuclear magnetic resonance probes

The conformation of cyclic 3′,5′-adenosine monophosphate in deuterium oxide has been determined at pH 2.0 and pH 5.5, using lanthanide ions as paramagnetic nuclear magnetic resonance probes.The lanthanide ion-induced shifts in the nuclear magnetic resonance energy for a given nucleus are dependent on the geometric position of that nucleus relative to the bound lanthanide ion. As expected, these shifts are pseudocontact in origin and are consistent with axial symmetry. Analysis of the concentration dependence of the shift shows that the lanthanide ion is bound to the phosphate entity giving a 1:1 complex. Further, base stacking and other intermolecular interactions are negligible.To confirm the conformation, which is found from a computer search with the above shift data, we have measured the changes in relaxation times, T₁ and T₂, induced by binding of Gd³⁺. The geometric dependence of these relaxation effects is different from that of shifts, being dependent only on distance. The agreement of these data with the computer “shift” conformation is satisfactory.Some ³¹P nuclear magnetic resonance experiments were done to confirm the metal co-ordination position although, here, there are contact contributions to both shift and relaxation.The computer program finds the conformations that have the correct geometry to account for the shift data, by searching all possible conformations. Non-bond rotations were used as a method of changing the pucker of the phosphate and ribose rings, the position of the base being defined by a single bond rotation. The nuclear magnetic resonance data and minimum van der Waals' distances were used as “active filters” in the computer search.At both values of the pH we have found closely related families of solutions, with the pucker of the phosphate and ribose rings roughly similar to those in an approximate X-ray study of cyclic AMP. The orientation of the base varies with pH.     

Equilibrium studies on neutral red-DNA binding.

Spectrophotometric binding studies were undertaken on the interaction of neutral red with native and heat-denatured, sonicated, calf thymus DNA in a 0.2M ionic strength buffer containing Tris–sodium acetate–potassium chloride at 25°C. The pK_A of neutral red was found to be 6.81. At pH 5 the binding of protonated neutral red was complicated even at low concentration ratios of dye to DNA. In the pH range 7.5–8.5 the tight binding process could be studied and it was found that both protonated and free base species of neutral red significantly bind with DNA having association constants (in terms of polynucleotide phosphate) of 5.99 × 10³ M^?1 and 0.136 × 10³ M^?1, respectively, for native DNA and 7.48 × 10³ M^?1 and 0.938 × 10³ M^?1, respectively, for denatured DNA. The pK_A value of the neutral red–DNA complexes were 8.46 for native DNA and 7.72 for denatured DNA. These results are discussed in terms of possible binding mechanisms.     

Nuclear magnetic resonance study of the impact of ribose 2′-O-methylation on the aqueous solution conformation of cytidylyl-(3′ → 5′)-cytidine

In order to obtain information about the conformational characteristics at the nearestneighbor level in the 2′-O-methylated region of t-RNA, as well as in the bizarre 5′-terminus of eucaryotic mRNA, a detailed nuclear magnetic resonance study of 2′-O-methyl-cytidylyl-(3′ → 5′)-cytidine (CmpC) was conducted. Proton spectra were recorded at 270 MHz in the Fourier mode in D₂O solutions, 0.01M, pD 7.3 in the temperature range 5–80°C. Complete accurate sets of nmr parameters were derived for each of the nucleotidyl units by a combination of homo-nuclear decouplings and simulation iteration methods. The data were translated into conformational parameters using procedures developed in earlier studies from these laboratories. It is shown that the ribofuranose ring exists at a ²E ? ³E equilibrium with clear preference [(75–80)%] for the ³E mode. The C(4′)-C(5′) and C(5′)-O(5′) bonds form a stable conformational network with outspoken preference for conformers in which Ψ₁, Ψ₂ ? 60° and ?₂ ? 180°. The orientation of the 3′-phosphate and 2′-O-methyl groups is such that ?₁′ ? 210° and ?″ ? 60°. The phosphodiester bonds are flexible and shift trends for base, H(1′), and H(5″) suggest the existence of a conformational blend of right-handed stack (g^?g^?), left-handed stack (g⁺g⁺), and unstacked arrays (tg^? and tg⁺). Elevation of temperature perturbs the ²E ? ³E equilibrium accompanied with modest depopulation of ψ₁, ψ₂ ? 60° and ?₂ ? 180° conformers. The major effect of elevation of temperature is in the increase of unstacked arrays at the expense of g^?g^? and g⁺g⁺ conformers. The shift trend of Cmp-H(3′) with temperature shows that torsional variation about O(3′)-P is facilitated by increase in temperature and the preferred rotamer about O(3′)-P in the unstacked form is t (ω₁′ = 180°). A detailed comparison of the aqueous solution conformations of CpC and CmpC reveals that 2′-O-methylation causes: (i) a reduction in the magnitude of _χ1; (ii) an increase in the population of ³E pucker at the 3′-nucleotidyl unit; and (iii) modest perturbations in the O(3′)-P and P-O(5′) bond conformations. Comparison of the aqueous solution conformations of AmpA and CmpC makes clear that the conformational properties of pyrimidine-pyrimidine and purine-purine dimers which carry a 2′-O-methylated 3′-nucleotidyl unit are significantly different.     

Studies on interaction between poly(L-lysine) and DNA of varied G + C contents

Interaction between polylysine and DNA's of varied G + C contents was studied using thermal denaturation and circular dichroism (CD). For each complex there is one melting band at a lower temperature t_m, corresponding to the helix–coil transition of free base pairs, and another band at a higher temperature t′_m, corresponding to the transition of polylysine-bound base pairs. For free base pairs, with natural DNA's and poly(dA-dT) a linear relation is observed between the t_m and the G + C content of the particular DNA used. This is not true with poly(dG)·poly(dC), which has a t_m about 20°C lower than the extrapolated value for DNA of 100% G + C. For polylysine-bound base pairs, a linear relation is also observed between the t′_m and the G + C content of natural DNA's but neither poly(dA-dT) nor poly(dG)·poly(dC) complexes follow this relationship. The dependence of melting temperature on composition, expressed as dt_m/dX_G·C, where X_G·C is the fraction of G·C pairs, is 60°C for free base pairs and only 21°C for polylysine-bound base pairs. This reduction in compositional dependence of T_m is similar to that observed for pure DNA in high ionic strength. Although the t′_m of polylysine-poly(dA-dT) is 9°C lower than the extrapolated value for 0% G + C in EDTA buffer, it is independent of ionic strength in the medium and is equal to the t_m⁰ extrapolated from the linear plot of t_m against log Na⁺. There is also a noticeable similarity in the CD spectra of polylysine· and polyarginine·DNA complexes, except for complexes with poly(dA-dT). The calculated CD spectrum of polylysine-bound poly(dA-dT) is substantially different from that of polyarginine-bound poly(dA-dT).     

The influences of water solvent on the structures and stabilities of Na+–AD conformers

The influences of water solvent on the structures and stabilities of the complex ion conformers formed by the coordination of alanine dipeptide (AD) and Na⁺ have been investigated using supramolecular and polarizable continuum solvation models at the level of B3LYP/6-311++G**, respectively; 12 monohydrated and 12 dihydrated structures of Na⁺–AD complex ion were obtained after full geometrical optimization. The results showed that H₂O molecules easily bind with Na⁺ of Na⁺–AD complex ion, forming an ion-lone pair interaction with the Na–O bond length of 2.1–2.3 Å. Besides, H₂O molecules also can form hydrogen bonds O_W–H_W···O(1), O_W–H_W···O(2), N(1)–H(1)···O_W or N(2)–H(2)···O_W with O or N groups of the Na⁺–AD backbone. The most stable gaseous bidentate conformer C7AB of Na⁺–AD is still the most stable one in the solvent of water. However, the structure of the most unstable gaseous conformer α′B of Na⁺–AD collapses under the attack of H₂O molecules and changes into C7AB conformation. Computations with IEFPCM solvation model of self-consistent reaction field theory give that aqueous C5A is more stable than C7_eqB and that the stabilization energies of water solvent on monodentate conformers of Na⁺–AD complex ion (about 272–294 kJ/mol) are more than those on bidentate ones (about 243 kJ/mol).