Martin
Dračínský
*ab,
Michal
Šála
b and
Paul
Hodgkinson
*a
aDepartment of Chemistry, Durham University, South Road, DH1 3LE, Durham, UK. E-mail: dracinsky@uochb.cas.cz; paul.hodgkinson@durham.ac.uk
bInstitute of Organic Chemistry and Biochemistry, Flemingovo nám. 2, 16610, Prague, Czech Republic
First published on 2nd June 2014
Nuclear magnetic resonance experiments, together with molecular dynamics simulations and NMR calculations, are used to investigate mobility of water molecules and sodium ions in solid hydrates of two nucleotides. The structure of guanosine monophosphate system (GMP) is relatively rigid, with a well-ordered solvation shell of the nucleotide, while the water molecules in the uridine monophosphate system (UMP) are shown to be remarkably mobile, even at −80 °C. The disorder of water molecules is observed in the 13C, 31P, and 23Na solid-state NMR experiments as multiple signals for equivalent sites of the nucleotide corresponding to different local arrangements of the solvation shell. Deuterium NMR spectra of the samples recrystallized from D2O also confirm differences in water mobility between the two systems. The experiments were complemented with NMR calculations on an ensemble of structures obtained from DFT molecular dynamics (MD) simulations. The MD simulations confirmed higher water mobility in the UMP system and the calculated chemical shifts and quadrupolar couplings were consistent with the experimental data. The disordered solvation shell in UMP is likely to be a good model for solvated nucleotides in general, with fast reorientation of water molecules and fluctuations in the hydrogen-bond network.
Because RNA and DNA are polyanions, their structure and biological function depends strongly on their association with metal ions. However, the dynamic non-covalent nature of these interactions has continually presented challenges to the development of accurate and quantitative descriptions.11 Metal ions are involved in almost every aspect of nucleic acid chemistry, ranging from neutralization of the anionic nucleic acids12 through specific stabilization of three-dimensional structures of NA molecules up to their effect as cofactors in RNA-mediated catalysis.13
Several spectroscopic methods have been employed in recent years to study the structures of hydrated ions and their aqueous environment.14 Although hydration patterns are difficult to study directly, techniques such as dielectric15 and terahertz16 spectroscopy, fluorescence labelling,17 optical spectroscopies,18,19 and nuclear magnetic resonance20 have revealed important information about the interaction of biomolecules with water in solutions, such as the number of water molecules in the first solvation shell, the affinity of water molecules for different groups, and the strength of the intermolecular hydrogen-bond interactions. However, the solvation shell dynamics are difficult to assess by these techniques.
The three-dimensional structure determinations of biological macromolecules, such as proteins and nucleic acids, by X-ray crystallography have significantly improved our understanding of biological processes. However, since it is very hard to identify hydrogen atoms accurately using X-ray diffraction, a detailed discussion of protonation and hydration sites can only be speculated upon so far.21 Direct detection of light alkali metal ions by diffraction techniques is also challenging, especially for sodium cations, because their X-ray scattering contributions are virtually identical to those of water, and Na+⋯O distances are only slightly shorter than strong hydrogen bonds between well-ordered water molecules.22 This often renders it impossible to identify Na+ ions, even with state-of-the-art diffraction techniques.
Here, we present a multinuclear NMR investigation of two hydrates of sodium salts of nucleotides (depicted in Fig. 1), namely disodium guanosine-5′-phosphate heptahydrate (GMP) and disodium uridine-5′-phosphate heptahydrate (UMP). These two systems represent typical examples of solid forms of nucleotides, which generally crystallise in the form of hydrates. Because of the high water content in the crystal lattice, they also represent a simple model of solvated nucleotides. Three crystal structures of GMP, all derived for data obtained at ambient temperature, have been deposited in Cambridge crystallographic structural database (CSD): GUOPNA01, GUOPNA10,23 and GUOPNA11.24 GUOPNA11 is re-analysis of GUOPNA01, where no hydrogen atoms were positioned. There are two non-equivalent molecules of GMP in the asymmetric unit (Z′ = 2). These have almost identical conformation, but differ in the arrangement of the surrounding solvent shell. The position of all water oxygen sites and sodium ions was determined by the X-ray analyses, and no disorder in the oxygen sites was observed. However, only 20 and 23 out of 28 water hydrogen atoms were located from a difference Fourier map in the GUOPNA10 and GUOPNA11 structures, respectively. The structures of GUOPNA10 and GUOPNA11 differ in hydrogen positions, most importantly the ribose O2′–H orientation, leading to a slightly different hydrogen-bonding network in the two crystal structures (see Fig. S1 in ESI†). Because of the structural uncertainty, we redetermined the GMP structure at 120 K by X-ray diffraction. The 23Na MQMAS spectrum of GMP has been reported, and shows four spectral regions, consistent with the four distinct Na+ sites observed by X-ray analysis. Quadrupolar coupling constants and asymmetry parameters were extracted from the MQMAS spectra and assigned to individual Na+ sites on the basis of a simple correlation between CQ and the number and type of surrounding ligands.25 Similar tentative assignments of sodium signals have been done also for other sodium salts of nucleotides.26,27
In contrast, a high degree of disorder of sodium ions and water molecules was observed in the crystal structure of UMP (CSD code SURIPH10, Fig. 2).28 The nucleotide molecules form layers, and the disordered water molecules and sodium ions are located in channels between the UMP molecules, which are equivalent (Z′ = 1) according the X-ray analysis. This disordered aqueous layer might reasonably serve as a model of the solvation shell of nucleotides in solution.
Fig. 2 Left: crystal structure of UMP (SURIPH10 structure28) – water molecules and sodium ions are disordered in the channel formed by the nucleotide molecules; right: crystal structure of GMP determined in this work. |
By applying multiple NMR techniques, together with molecular dynamics (MD) and DFT calculations of NMR parameters, we investigate the nature of the disorder in the UMP crystal structure. Density functional theory molecular dynamics (DFT-MD) provides insight into the structure and dynamics of the nucleotide solvation shells. It has been demonstrated that DFT-MD simulations are superior to classical force-field simulations when modeling hydration shells of polar molecules; DFT-MD provided more structured solvation shells, and NMR calculations based on the DFT-MD simulations were in very good agreement with experiment.20,29,30 The combination of NMR experiments with theoretical calculations makes it possible to connect experimental observations with structural and dynamic characteristics of the studied systems.
Crystal data for GMP were collected at 120 K on an Agilent Gemini S-Ultra diffractometer (ω-scan, 0.5° per frame) yielding 9320 independent reflections (Rmerg = 0.1005). The structure was solved by direct method and refined by full-matrix least squares on F2 for all data using SHELXTL31 and OLEX2 (ref. 32) software. The crystal data and experimental details are summarized in the ESI.† All non-hydrogen atoms were refined with anisotropic displacement parameters. H-atoms were placed into the calculated (OLEX2) positions and refined in riding mode. Final wR2(F2) = 0.2118 for all data (609 refined parameters), conventional R1(F) = 0.0794 for 6470 reflections with I ≥ 2σ, GOF = 1.029. Crystallographic data for the structure have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication CCDC-987999.
High-resolution 2H, 13C, and 31P solid-state NMR spectra were obtained using a Varian/Chemagnetics InfinityPlus spectrometer operating at 76.7 MHz for 2H, 125.7 MHz for 13C, 202.3 MHz for 31P, and 499.7 MHz for 1H and/or a Bruker Avance III spectrometer operating at the same frequencies. Samples were packed into 4 or 5 mm magic angle spinning rotors and measurements taken using a MAS rate of 10 kHz using cross polarisation (13C, 31P) or direct excitation (2H). No correction was made to the set temperatures to correct for the frictional heating of the sample under sample spinning. Variable temperature experiments were done at low temperatures first, to minimise the dehydration of the samples. 1H and 2H T1 (spin–lattice) relaxation times were determined directly in 1H/2H spectra using a series of saturation recovery experiments with 10 kHz MAS (saturation used: 1000 π/2 pulses separated by 1 ms delay). While showing some interesting trends, the relaxation data could not be interpreted with great confidence; the results and discussion can be found in the ESI.†23Na MQMAS spectra were acquired using a Varian VNMRS 400 spectrometer operating at 105.78 MHz for 23Na. A two-pulse plus z-filter version of the experiment was used, with a 0.2 s recycle delay, 10 kHz spin rate and 256 increments in the indirectly detected dimension. The typical CP conditions used were: recycle delay 4 s, contact time 2 ms, acquisition time 40 ms. Carbon-13 spectra were referenced with respect to external neat tetramethylsilane by setting the high-frequency signal from a replacement sample of adamantane to 38.5 ppm. Phosphorus spectra were referenced with brushite (δ = 1 ppm), deuterium spectra with D2O (δ = 4.8 ppm), and sodium spectra with solid NaCl (δ = 0 ppm). Spinning sideband analysis using iterative fitting of the full 2H and 31P spectra was performed in Gsim33 and pNMRsim34 programs.
The NMR tensors of the infinite crystals were calculated by the CASTEP program,35 version 6.0, which is a DFT-based code. Electron-correlation effects were modeled using the generalized gradient approximation of Perdew, Burke, and Ernzerhof.36 For the atomic position optimization, we employed ‘on-the-fly’ pseudopotentials, a planewave cutoff energy of 600 eV, with integrals taken over the Brillouin zone using a Monkhorst–Pack37 grid with a minimum k-point sampling of 0.05 Å−1. The NMR calculations were performed using the GIPAW approach38,39 at a larger cutoff of 900 eV.
To better understand the mobility of the solvation shells in both structures, molecular dynamics (MD) was performed at high temperature, followed by optimizations of geometry snapshots from the MD runs. Our X-ray diffraction structure of GMP was used as the starting structure for the MD simulation of this system. The SURIPH10 structure from CSD was used for UMP, after manually adding sodium ions and water molecules into the positions with the highest residual electron density, and consistent with sodium ions being five- or six-coordinated to water or nucleotide oxygens. The starting UMP structure (depicted in Fig. S2, fractional coordinates in ESI†) was then optimised. This partially random generation of the initial structure does not necessarily lead to the global energy minimum, but the potential energy equilibrated quickly at the beginning of the MD simulation, which indicates that the structure was suitable for the simulation. Born–Oppenheimer molecular dynamics (BOMD) simulations were run in CASTEP using an NVT ensemble maintained at a constant temperature of 1000 K using a Langevin thermostat, a 0.5 fs integration time step, ultrasoft pseudopotentials,40 a planewave cutoff energy of 300 eV, and with integrals taken over the Brillouin zone using a Monkhorst–Pack37 grid of a minimum k-point sampling of 0.1 Å−1. The MD parameters were tested with respect to the convergence of atomic forces; an increase of the cutoff energy or k-point sampling led to changes in the calculated forces lower than 0.1 eV Å−1. The lattice parameters and the positions of the nucleotide molecules were fixed to the experimental values, and so no dispersion corrections were required to maintain cell volumes during the MD simulations. No symmetry constraints were applied during the runs. Simulation runs of 5 ps were performed for both compounds. 23 geometries were selected at 0.6, 0.8, 1.0…5.0 ps of each run for geometry optimisation. The five lowest energy optimised UMP structures (differing by less than 1 eV per unit cell from the lowest energy) were used for NMR calculations (see Fig. S3 in the ESI†).
Fig. 3 (a) Variable temperature 31P CP-MAS NMR spectra of GMP. The signal at 5 ppm belongs to the dehydrated form. (b) Variable temperature 31P CP-MAS NMR spectra of UMP. The dashed lines are guides for the eye to follow the temperature changes. Centre-bands only are shown; complete spectra are in the ESI† (Fig. S6). |
On the other hand, much more complicated 31P NMR spectra were observed for UMP. X-ray crystallography determined Z′ to be 1, which should lead to a single phosphorus signal in the spectra, but at −40 °C we observed a broad distribution of phosphorus signals between 1 and 6.5 ppm. With increasing temperature, the spectrum becomes narrower, but at least seven phosphorus sites can still be distinguished at 40 °C. The spectral narrowing is reversible, but the sample dehydrates irreversibly at temperatures above 40 °C. The observation of more than one phosphorus signal may be explained by the disorder of the water molecules and sodium ions in the channels between the UMP molecules; different positions and orientations of the water and sodium will influence the phosphorus chemical shift of the phosphate group and individual signals for each geometry will be observed if the rate of exchange between different local geometries is slow. The relative intensities of the spinning side bands were similar to those of the GMP crystals, i.e. the chemical shift anisotropy is similar for all phosphorus sites in both samples.
13C cross-polarization (CP) magic-angle spinning (MAS) NMR is an efficient method for crystal structure characterization, including determining Z′ by means of comparing the number of observed resonances with the number of nonequivalent carbon atoms present in the molecule.42,43 Similarly to the 31P spectra, two sets of signals corresponding to the two non-equivalent GMP molecules accompanied by one set of signals of the dehydrated form were observed in the 13C NMR spectra of GMP in a broad temperature range (−80–40 °C), and a distribution of signals was observed in carbon spectra of UMP (Fig. 4 and S7†). The UMP signals start to merge into one set of signals at 40 °C, but dehydration occurs at higher temperatures. It is likely that the spectrum at 40 °C is close to the fast exchange limit. However, the narrow range of NMR frequencies spanned by a given site (of the order of 200 Hz) means that the exchange rates are low in absolute terms, and the barriers involved must, therefore, be relatively high. It is, therefore, unsurprising that the fast-exchange limit is not reached for all the 31P resonances at 40 °C, Fig. 3(b), since these span a significantly larger range of frequencies (about 1200 Hz). It is clear from the 31P spectrum, however, that multiple sites and effective barriers are involved, with the fast exchange regime for different sites being reached at different temperatures.
Fig. 4 Variable temperature 13C CP-MAS spectra (ribose region) of (a) GMP (dashed lines indicate the dehydrated form) and (b) UMP. See Fig. 1 for atom numbering. The aromatic regions of the spectra are shown in the ESI (Fig. S7). |
The sodium MQMAS spectrum of GMP at −70 °C (Fig. 5) is similar to the spectrum reported previously.25 At room temperature the signals broaden and start to merge indicating that the sodium ions may be able to exchange their positions in the crystal structure, although there is also likely be broadening associated with relatively slow dynamics on the order of the MAS frequency (10 kHz).44,45 Again, the 23Na MQMAS spectrum of UMP exhibits more signals than the two expected from the crystal structure, and broadening of the signals is apparent at room temperature. It is noticeable, however, that the room temperature spectrum shows evidence of two broad features, although the highly disordered nature of the Na environments observed by XRD means these cannot be linked in an obvious way to the crystal structure.
Fig. 5 23Na MQMAS spectra of UMP and GMP; the signals of the dehydrated form of GMP are indicated by dashed circles and arrows. |
Some care is required when interpreting the deuterium spectra of the nucleotides crystallised from D2O since these also contain signals from exchangeable protons i.e. 2 ribose–OH groups and 3 (GMP) or 1 (UMP) amine/amide hydrogens. Moreover many, if not all, of these sites will be undergoing chemical exchange via the hydrogen bonding network. The dominant signal in the 2H MAS spectrum of UMP–D2O is remarkably narrow (only a few spinning side bands are observed), and decreasing the temperature to −80 °C does not lead to significant changes. This is most likely to be a single signal arising from the water + ribose OH hydrogens; there is evidence, see Fig. S8,† of a weak shoulder around 10 ppm which can be tentatively assigned to the uridine NH. It is likely that the exchange between the 14 water and 2 ribose hydrogens is sufficiently fast (compared to the quadrupolar interactions) for a single averaged set of quadrupolar parameters (dominated by those of the water) to be observed. The experimental parameters obtained by spectral fitting (Fig. 6) are CQ = 35 ± 1 kHz, η = 0.52 ± 0.10 at 22 °C, and CQ = 38 ± 1 kHz and η = 0.25 ± 0.10 at −80 °C. Typical values of the 2H quadrupolar coupling constant in various “rigid” solids varies from 160 to 340 kHz with 0 ≤ η ≤ 0.15 and the spectra thus indicate that the water molecules in UMP–D2O are highly mobile over the whole temperature range and the rate of water reorientation is on the order of hundreds of kHz or higher.46 The degree of dynamic disorder at −80 °C is surprising; only a modest broadening associated with some slowing of dynamics is observed. Presumably the water molecules are effectively in a metastable state, with multiple low energy barriers, and are unable to settle into a well-defined energetic minimum. The GMP–D2O sample proved difficult to obtain and sensitive to dehydration; the partial results obtained are presented in Fig. S9† and associated discussion in the ESI.†
Fig. 6 Experimental 2H MAS spectra (MAS rate 10 kHz) of UMP at 20 °C and −80 °C (grey solid lines) and simulated spectra (black dashed lines). |
In the case of GMP, all the generated structures had a similar arrangement of the sodium ions and water molecules, including the overall hydrogen-bonding network (see Fig. 7), which means that the solvation shell lies in a deep potential energy well and no other local energy minima were accessed by the MD simulation. On the other hand, the generated UMP structures differ significantly, with sodium ions and water molecules occupying different positions in the channels formed by the nucleotide molecule.
In the course of the MD simulations of UMP, we observed continuous reorientation of water molecules leading to different structures of the hydrogen-bond network in the geometry snapshots. Occasionally, individual water molecules moved between coordination spheres of the surrounding sodium ions, i.e. the shortest Na⋯O distance connected a given water molecule with different sodium atoms in the course of the simulation. However, the simulations were not sufficiently long for individual water molecules to explore the whole space in the nucleotides' channels; the average RMSD of the water oxygen and sodium atom positions from the starting structure was 2.11 and 1.96 Å, respectively (compared to the much smaller RMSDs of 0.88 and 0.91 Å, respectively, observed for the GMP system).
The limited length of the MD simulations may lead to insufficient sampling of the configurational space and other low energy minima might be missing in the ensemble, and the unphysical nature of the simulations limit the scope for quantitative analysis. We can, however, clearly see differences in the dynamic behaviour of the two systems, which reflect the results of the diffraction experiments, i.e. the solvation shell in the GMP crystal is regularly ordered, whereas sodium atoms and water molecules in the UMP crystal can be found in different arrangements, leading to the inability of the diffraction experiments to describe the UMP solvation shell.
Sodium site | Na coordinationa | C Q,calc/MHz | C Q,exp/MHzb |
---|---|---|---|
a W – water molecule; OH – ribose hydroxyl group; N – guanine nitrogen atom N7. b The experimental values are from ref. 25 (uncertainties not given), but our assignment based on CASTEP calculations interchanges the quadrupolar couplings of Na3 and Na4. | |||
Na1 | 4W,2OH | 1.44 | 1.30 |
Na2 | 6W | 1.67 | 1.85 |
Na3 | 6W | 2.27 | 2.30 |
Na4 | 4W,2N | 1.75 | 1.85 |
We also calculated NMR parameters for the five lowest energy optimised geometry snapshots from the MD simulations of UMP. The limited length of the MD simulation and the number of NMR calculations is partly compensated by the size of the unit cell (eight nucleotide molecules, 16 sodium ions, and 56 water molecules). No symmetry constraints were applied during the simulations, therefore the structure of the solvation shell of the eight nucleotide molecules differed, and so the nucleotide 31P NMR parameters were averaged for 40 local solvation arrangements. Importantly, the NMR data (span of chemical shifts and averaged quadrupolar couplings) calculated for the individual optimised snapshot geometries are qualitatively consistent with the results averaged for the five structures. For example, the calculated phosphorus chemical shifts are distributed over similar ranges in the five snapshots (see Table S1 in ESI†), i.e. increasing the number of structures for NMR calculation is unlikely to change the qualitative picture significantly. We do not see any clear correlation between the calculated phosphorus chemical shifts and the hydration pattern (number of hydrogen bonds or number of sodium ions in close vicinity) of the phosphate group.
The calculated phosphorus chemical shift anisotropy is found in the range of 55–66 ppm and is almost unaffected by the dynamics, i.e. the shielding tensor of one phosphorus site averaged over the five optimised structures is very close to the individual CSA tensors. Carbon chemical shifts of individual sites were distributed over 2.8–9.0 ppm with the narrowest distribution for carbon C4, which had also the narrowest line in experimental spectra, and the broadest distribution for carbon atoms C5 and C6, which have also the broadest experimental signal (see Fig. S7 in ESI†). The convergence of the carbon chemical shift spans with the number of geometry snapshots is illustrated in Fig. S10.†
The calculated deuterium quadrupolar couplings of the water hydrogens in the five optimised UMP structures can be found in a broad range 130–290 kHz, which is much more than the experimental value of 35–38 kHz. As we have previously demonstrated, fast vibrational/librational motion in “rigid” solids leads to reduction of chemical shift anisotropy and quadrupolar couplings by only a few per cent and cannot thus explain this difference between calculated and experimental deuterium quadrupolar couplings.48 When electric field gradient tensors of the hydrogen atoms in individual water molecules are averaged (i.e. assuming fast C2 jumps of water), the quadrupolar couplings of the 56 nonequivalent water molecules are reduced to ca. 100–150 kHz. On the other hand, when the electric field gradient tensors of all water hydrogen atoms in each of the five individual optimised structures are averaged, the resulting range of deuterium quadrupolar coupling constants is 25–31 kHz (η = 0.07–0.47), which agrees much better with the experimental value. This agreement confirms the high mobility of the water molecules. When EFG tensors of the ribose OH hydrogens are averaged together with all water protons (assuming that site exchange is fast compared to the quadrupolar couplings), the calculated quadrupolar couplings are reduced by further ca. 10 kHz.
The experimental sodium spectra of UMP are too complex to compare with the calculations directly, and there was no evidence in the calculation results of two average Na environments that appear to be observed in the room temperature MQMAS spectrum. It is likely that the CASTEP calculations are not sufficiently robust for 23Na shifts to try to explain rather subtle features of the experiment, because the shift range is quite narrow. From the inspection of the MQMAS spectra it can be expected that the experimental sodium quadrupolar couplings will be of the same order as in the case of GMP, i.e. a few MHz. When calculated EFG tensors of the individual sodium sites are averaged for the five UMP structures, the resulting 16 sodium quadrupolar couplings are in the range 0.9–2.4 MHz, which is in a qualitative agreement with the experiment. On the other hand, when the EFG tensors of all sodium sites are averaged, the calculated quadrupolar coupling is close to 0.4 MHz, which would lead to much narrower sodium spectra. This is in agreement with the distribution of chemical shifts observed experimentally.
The presented NMR experiments prove major differences in the dynamics of the solvation shell in the two studied systems. Water molecules and sodium ions are relatively rigid in the GMP structure, while they are very mobile in the UMP system, even at temperatures far below 0 °C. The disordered channels in the UMP crystals could be described as a rubbery state, with increasing thermal energy turning previously static disorder into dynamic disorder. The variable temperature NMR spectra of the UMP system exhibit continuous changes of the spectral parameters (chemical shifts, relaxation times, and quadrupolar coupling constants), which confirms the existence of multiple local arrangements of the UMP solvation with a slow interconversion between them. The energy barriers involved in the interconversions have probably a broad distribution; different energy barriers are overcome at different temperatures leading to continuous changes of the spectra.
The differences in the dynamic behaviour of the two systems are further confirmed by the MD simulations and NMR calculations. The structure of the GMP solvation layer was stable, even at the unphysical MD simulation at 1000 K. On the other hand, the MD simulation of the UMP system led to continuous changes in the hydrogen-bond network and the structure of the solvation shell. The spans of carbon and phosphorus chemical shifts calculated for the ensemble of structures exctracted from the UMP simulation were in qualitative agreement with the experiments. The short time of the simulation did not allow the water molecules to explore the whole configurational space, but when the calculated deuterium electric field gradient tensors of all water hydrogen atoms were averaged, the resulting deuterium quadrupolar coupling agreed well with the experimental value.
The method of generation of the ensemble of structures of disordered solvation shell by molecular dynamics simulation at high temperature within a fixed host structure, is a straightforward way of finding different local minima on the potential energy hypersurface (similar to simulated annealing). However, the MD method cannot be used for a quantitative interpretation of the dynamics of the system, because the simulations were performed at unphysical conditions.
The disordered solvation shell in UMP is probably close to the situation in solution with fast reorientation of water molecules and fluctuations in the hydrogen-bond network. The combination of X-ray diffraction and NMR experiments with MD simulations and calculations of NMR parameters can thus provide detailed information on the solvation shell dynamics on a molecular level.
Footnote |
† Electronic supplementary information (ESI) available. CCDC 987999. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c4ce00727a |
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