Structure and energy of nucleic acid base–amino acid complexes: 1. 1-methyl-uracil-acrylamide

Journal of Molecular Structure 478 (1999) 155–162

Structure and energy of nucleic acid base–amino acid complexes: 1. 1-methyl-uracil-acrylamide I. Galetich b, M. Kosevich b, V. Shelkovsky b, S.G. Stepanian b, Yu.P. Blagoi b, L. Adamowicz a,* b

a Department of Chemistry, University of Arizona,Tucson, Arizona 85721,USA Institute for Low Temperature Physics and Engineering, National Academy of Sciences of Ukraine, 47 Lenin Avenue,Kharkov 310164,Ukraine

Received 1 September 1998; accepted 12 October 1998

Abstract The temperature-dependent field ionization mass spectrometry method was applied to determine the interaction energy between 1-methyluracil and acrylamide, which mimics the side chains of the natural aminoacids Asparagine and Glutamine. The experimental enthalpy of formation of 1-methyluracil-acrylamide dimer derived from Vant-Hoff plots is ⫺ 40.6 ^ 4.2 kJ mol ⫺1. Two equilibrium geometry configurations of the 1-methyluracil-acrylamide dimer stabilized by intermolecular N– H…O H-bonds were found via quantum-chemical calculations at the DFT/B3LYP /6-31⫹⫹G** and MP2/6-31⫹G* levels of theory. The two methods yielded similar interaction energies for the two configurations, which are in good agreement with the experimental result. 䉷 1999 Elsevier Science B.V. All rights reserved. Keywords: Amino acid; 1-methyl-uracil-acrylamide

1. Introduction Interaction between proteins and nucleic acids occurs at all stages of replication and expression of DNA and in many processes of bio-regulation. For example, the interactions between nucleic acid bases and the amide group via intermolecular H-bonds were directly identified in the X-ray study of structure of a specific ‘repressor-operator’ complex of bacteriophage 434 [1]. Smolyaninova et al. studied the DNA-protein interactions and demonstrated that aminoacids Asparagine and Glutamine destabilize the DNA by forming H-bonds between their amide groups and nucleic acid bases in single strand DNA [2]. The H-bonding interaction between the * Corresponding author; e-mail: [email protected]

aminoacid amide group and nucleic acid bases was also investigated in solvent chloroform with the use of the NMR spectroscopy method [3]. Both studies were related to the important role of the amide group as a part of protein structure, which is involved in untwisting of the DNA double helix. Our knowledge of the mechanisms of the DNAprotein interactions is still very limited. Mainly the large sizes of the systems involved and the complexity of the interaction effects cause this. The approach, which we have taken in, our studies were to first investigate some simpler model systems, which contain some characteristic features of DNA-protein complexes in order to eventually move towards more complex and realistic models. The main goal of these model experiments is to establish a detailed molecular mechanism of the specific recognition of nucleic acid

0022-2860/99/$ - see front matter 䉷 1999 Elsevier Science B.V. All rights reserved. PII: S0022-286 0(98)00756-X

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bases by the side chains of amino acids. Study of the interactions between the components of nucleic acids and protein fragments allows one to describe the role of different atomic groups in formation of protein– nucleic acid complexes and to predict changes in their stability, caused by the changes in the primary structure of the biopolymers. A method of temperature-dependent field ionization (TD FI) mass spectrometry were successfully applied in experimental determination of the energy of formation of pair and triple complexes of nitrogen bases with water molecules in vacuum [4–9]. This technique is one of a few methods whose results can be directly compared with the theoretical data obtained by quantum chemical calculations in the so-called ‘vacuum approximation’. The aim of the present series of work is to elucidate the molecular specificity of the interaction between nucleic acid bases and side chains by means of experimental measurement of enthalpy of formation of hydrogen-bonded complexes using TD FI mass spectrometry and by means of theoretical calculations of energy and structure of these associates using ab initio and DFT methods. More specifically, in the present work we consider the structural and energy parameters of the complex of 1-methyluracil (m 1Ura) with acrylamide (Acr). This system represents a model of interaction between the amide group of amino acids with the functional groups of the Uracil molecule which are free and open to interaction in the case of helical and unfolded states of nucleic acids.

2. Experimental method A method of temperature-dependent field ionization mass spectrometry is based on the ionization of molecules in a high heterogeneous electric field whose strength is of the order of 10 10 V/m. In such a field near a positively charged surface of the metal emitter, a transition of a valence electron from the analyzed molecule to the metal of the FI emitter can occur the tunnel mechanism without noticeable excitation of the molecule (so-called ‘soft’ ionization conditions) [10,11]. As a result of low excitation energy gained by the molecular ions in the ionization process, the FI mass spectrum contains, as a rule, a high abundance of the molecular ions M ⫹ and a

relatively low abundance of fragment ions. At the same time, the heterogeneity of the electric field evokes attraction of the molecules to the needle of the emitter, which leads to the increased flux of the molecules towards the surface. As a result, a localized region of the increased concentration is created near the emitter surface, where the molecules have a possibility to bear multiple collisions, leading to formation of neutral complexes. The above-mentioned soft character of the ionization process allows one to obtain ions of the intact dimers and trimers of small organic molecules stabilized by week non-valence van-derWaals interactions and hydrogen bonds [4,9,10]. Under the standard experimental conditions at the field strength of (0.2–1) × 10 10 V/m, the state of the system is determined by the dynamic equilibrium between the entering of the neutral particles from the bulk volume to the ionization region and the moving of the ions and non-ionized neutrals back to the volume. The relative concentration of the molecular and cluster ions corresponds to the concentrations of the initial components and the final products of the dimerization and trimerization reactions. These concentrations are registered mass-spectrometrically during extended sampling time (usually several hours) as stable ion currents at the corresponding mass values. Analysis of the thermodynamic equilibrium of the association reactions of nitrogen bases under the conditions of mass spectrometric experiment [4] showed that the time of ionization t i, which corresponds to the molecule lifetimes in the neutral state prior to their ionization, during which they can undergo multiple interactions with each other and the emitter surface with efficient thermalization, is of the order of 10 ⫺6 s. At the same time, the average time span between the collisions t 0 is 10 ⫺11 s. An ion formed because of FI is removed from the region immediate to the emitter surface during t 00 10 ⫺12 – 10 ⫺13 s. The relation, t i p t 0 ⬍ t 00 , points to a low probability of collisions of the ionized particles with the neutral molecules, which allows one to exclude ion–molecule reactions from the consideration. The existence of the thermodynamic equilibrium in the system under study (at the constant parameters of the mass spectrometric experiment) is confirmed by the stability of the association constant, Kass, determined on the basis of the ratio of the abundance of

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0–1.2 A and measured by copper–constantan thermocouple attached near the point of the needle welding to its holder. The stepwise change of emitter temperature caused sufficiently rapid (in 1–1.5 min) achievement of the temperature plateau, resulting in a shift of the equilibrium of the association reaction and in a redistribution of the abundance of the ions of the monomers and dimers. In the present work, samples of m 1Ura and acrylamide purchased from ‘Reanal’, Hungary, were used.

3. Theoretical methods

Fig. 1. Field ionization mass spectrum of the mixture of acrylamide (Acr) and 1-methyluracil (m 1U). Emitter temperature Te ˆ 20⬚C, Emitter potential: Ue ˆ ⫹ 5 kV. Peaks at one mas unit higher than the molecular ion are a superposition of 13C isotope-containing and protonated species.

the reacting molecules and their associates in the FI mass spectra. Mass spectrometric experiments were carried out with the help of a magnetic sector mass spectrometer-MI-1201 (‘Electron’ Works, Sumy, Ukraine) equipped with a FI ion source. The set-up is described elsewhere [5,9]. The main element of the ion source, which creates the high electric field, is the FI emitter. The emitter is a single tungsten needle, whose tip has a radii (re), as estimated by electron microscopy [12], ˚ . The needles were prepared from of about 4000 A tungsten rods of 0.15 mm in diameter by electrolytic etching [13]. At standard conditions, the emitter potential, Ue, was ⫹ 5 kV, which provided the electric field strength, estimated as Fe 艑 Ue/5re ˆ 0.25 × 10 10 V [10,11], sufficient for an efficient ionization of the analyte and the for production of stable longlasting ion currents. The introduction of compounds under study to the interaction zone near the emitter was performed using two glass evaporators with the volume of 0.5 m 3 each and with a hole of diameter 0.1 mm. The heating of the evaporators containing crystals of the analytes provided controlled and stable molecular fluxes in a wide density range. To obtain temperature-dependencies, the temperature of the emitter was controlled by resistive heating of the emitter metal holder using electric currents with ie ˆ

Structures and interaction energies of the possible Acr–m 1Ura dimers were calculated at the DFT and MP2 [14,15] levels of theory. Firstly, the dimer geometries were fully optimized at the DFT/631⫹⫹G** level. The three-parameter density functional designated as B3LYP was used in the calculations. It includes Becke’s gradient exchange correction [16], the Lee et al. correlation functional [17] and the Vosko, Wilk, Nusiar correlation functional [18]. The calculations converged to two almost planar Acr–m 1Ura dimers stabilized by N–H…O H-bonds. For the dimer configurations found at the DFT level, we calculated harmonic frequencies to evaluate the ZPVE corrections to the dimer formation energies. Next, we reoptimized the dimer geometries at the MP2/6-31⫹G* level of theory and calculated the dimer formation energies at that level. The interaction energies of the dimers, both at the DFT and MP2 levels, were calculated with the account of the basis set superposition error (BSSE) by the counterpoise method of Boys and Bernardi [19]. The method involves a single calculation for the dimer and two calculations for the monomers with the basis set of the dimer. In the calculations of the monomers, the monomer equilibrium geometries were used. The BSSE-corrected interaction energy was calculated for each dimer as the difference of the dimer energy and the monomer energies obtained with the dimer basis set. The BSSE corrections for the monomers were calculated as energy differences between the monomer energies calculated in the monomer and dimer basis sets at the equilibrium geometries of the monomers. Additionally, the MP2/ 6-31⫹⫹G** single point calculations for the two

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the intensities of the peaks in the FI mass spectra are related to the concentrations of the molecules in the ionization zone through the following relation: Ix …F† ˆ sx …F†·nx …F†;

…1†

where Ix(F) is intensity of the peak of the molecule X at the field strength value F; s x(F) is the ionization coefficient; and nx(F) are the concentrations of the neutral molecules. In the case of the m 1Ura– Acr dimer, the relative constant of association, Kass, is: Kass ˆ nAcr⫺m1 Ura =…nAcr nm1 Ura † Fig. 2. Dependences of the ion currents (I) vs currentie of the emitter resistive heating for acrylamide (1), 1-methyluracil (2) and acrylamide -1-methyluracil dimers (3).

dimers were carried out using geometries optimized at the MP2/6-31⫹G* level of theory. All calculations presented in this work were performed on IBM RS6000 workstations using the GAUSSIAN 94 software package [20].

4. Results and discussion A typical FI mass spectrum of the mixture of m 1Ura and acrylamide (Fig. 1) contains signals corresponding to molecular ions of the initial components and signals corresponding to the dimers, m 1Ura– m 1Ura, Acr–Acr and Acr– m 1Ura, stabilized by hydrogen bonds. The temperature-dependencies of the ion currents of the monomers and of the heterodimer are presented in Fig. 2. The values of

Fig. 3. Temperature dependence of Kass for acrylamide -1-methyluracil dimers.

ˆ IAcr⫺m1 Ura =…IAcr Im1 Ura †·s*…F†;

…2†

where s * (F) is the effective ionization coefficient. The real constant of the chemical equilibrium, K 0 ass, and the relative association constant Kass, are related via ratio: Kass ˆ K 0ass :s*…F†:

…3†

It was shown [4,11] that at constant experimental conditions and when the studied molecules have similar ionization potentials the temperature-dependence of s *(F) could be neglected. The association constant, Kass, was calculated in each point of the dependency (lgI-f(ie)). Temperature-dependencies of Kass, calculated as lnKass ˆ ⫺…DH=…RT†† ⫹ const:;

…4†

allowed construction of the Vant–Hoff plots (Fig. 3) and determination of the enthalpy of formation of the hydrogen-bonded complex Acr–m 1Ura. The enthalpy value DH derived from this analysis is ⫺40.6 ^ 4.2 kJ mol ⫺1. As mentioned previously, two planar structures of the Acr–m 1Ura dimer stabilized by inter-molecular N–H…O H-bonds were determined from the calculations. The equilibrium structures found, that we label Dimer A and Dimer B, are depicted in Fig. 4. The main difference between the two dimers is in which of the uracil oxygen atoms acts as the H-bond proton acceptor. It should be noted that the DFT/B3LYP/631⫹⫹G** method predicts shorter lengths of the Hbonds comparing to the MP2/6-31⫹G* method: ˚ (H9…O22) at the DFT ˚ (O8…H20), 1.820 A 1.906 A ˚ level and 1.917, 1.840 A for the corresponding Hbonds at the MP2 level.

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Fig. 4. Equilibrium Dimer A (top) and Dimer B (bottom) geometries calculated at the MP2/6-31 ⫹ G* level. (The geometries of the dimers are available from the corresponding author.)

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Table 1 Calculated at the DFT/B3LYP/6-31⫹⫹G*** level of theory, energies (a.u.), interaction energies (IE, kJ mol ⫺1) and zero-point vibration energies (ZPVE, a.u.) for the Acr-m 1Ura dimers, together with BSSE and ZPVE corrected interaction energies. all the energies were obtained for the dimer geometries optimized at the DFT/B3LYP/6-31⫹⫹G** level

Energy IE ZPVE a ZPVE correction BSSE correction b Acr m 1Ura Total IE (BSSE corrected) IE (BSSE and ZPVE correected) a b

Dimer A

Dimer B

⫺701.4986007 ⫺52.32 0.1859309 0.0010486

⫺701.4966400 ⫺47.17 0.1857862 0.0009761

0.0004226 0.0004803 0.0009029 ⫺49.95 ⫺52.70

0.0004295 0.0004743 0.0009038 ⫺44.80 ⫺47.36

Calculated based on the DFT/B3LYP/6-31⫹⫹G* frequencies scaled by a factor of 0.95. Energy difference calculated for the equilibrium monomer geometries in the monomer and dimer basis sets.

Energies, zero-point vibrational energies, BSSEcorrections and interaction energies of the dimers calculated at the DFT/B3LYP/6-31⫹⫹G* and MP2/ 6-31⫹G* levels are presented in Tables 1 and 2, respectively. Analysis of the data allows one to draw the following conclusions: • Both methods predict that Dimer A is the most stable configuration, although the energy difference between the two dimers is small (⬍6 kJ mol ⫺1). • Non-BSSE-corrected interaction energies obtained at the DFT and MP2 levels are considerably

different, but the BSSE-corrected results are almost identical. • Accounting for the BSSE considerably improves agreement with the experimental interaction energy, especially for the MP2 method. On other hand, the ZPVE correction leads to an increase of the calculated interaction energies, but the agreement between predicted and experimental energies still remains good. Analyzing the results presented in Tables 1 and 2, we should note that the BSSE corrections are significantly different for the two methods used in

Table 2 2Calculated energies (a.u.), interaction energies (IE, kJ mol ⫺1) and zero-point vibration energies (ZPBE, a.u.) for the Acr–m 1Ura dimers, together with BSSE and ZPVE corrected interaction energies. All the energies except the ZPVE were obtained for the dimer geometries optimized at the MP2/6-31⫹G* level

Energy IE ZPVE a ZPVE correction BSSE correction b Acr m 1Ura Total IE (BSSE corrected) IE (BSSE and ZPVE corrected) a b

Dimer A

Dimer B

⫺699.4007896 ⫺62.04 0.1859309 0.0010486

⫺699.3994032 ⫺58.40 0.1857862 0.0009761

0.0028586 0.0022317 0.0050903 ⫺48.68 ⫺51.43

0.0026273 0.0023683 0.0049956 ⫺45.28 ⫺47.84

Calculated based on the DFT/B3LYP/6-31⫹⫹G** frequencies scaled by a factor of 0.95. Energy difference calculated for the equilibrium monomer geometries in the monomer and dimer basis sets.

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Table 3 Calculated energies (a.u.), interaction energies (IE, kJ mol ⫺1) and zero-point vibration energies (ZPVE, a.u.) for the Acr–m 1 Ura dimers, together with BSSE and ZPVE corrected interaction energies. All the energies except the ZPVE were obtained at the MP2/6-31⫹⫹G** level for the dimer geometries fully optimized at the MP2/6-31⫹G* level.

Energy IE ZPVE a ZPVE correction BSSE correction b Acr m 1Ura Total IE (BSSE corrected) IE (BSSE and ZPVE corrected) a b

Dimer A

Dimer B

⫺699.491107 ⫺60.89 0.1859309 0.0010486

⫺699.4895491 ⫺56.80 0.1857862 0.0009761

0.0025172 0.0020328 0.0045490 ⫺48.95 ⫺51.70

0.0021942 0.0021865 0.0043807 ⫺45.30 ⫺47.86

Calculated based on the DFT/B3LYP/6-31⫹⫹G** frequencies scaled by a factor of 0.95. Energy difference calculated for the equilibrium monomer geometries in the monomer and dimer basis sets.

the calculations: 2.37 kJ mol ⫺1 for both dimers at the DFT level and 12.97 (Dimer A) and 13.12 kJ mol ⫺1 (Dimer B) at the MP2 level. It is not clear whether the differences is because of the different basis sets used for DFT and MP2 calculations, or because of the fact that the dimer geometries obtained by the methods differ in some parameters, or it is related to the nature of the methods. To elucidate this question, we performed additional calculations at the MP2/631⫹⫹G** level for the dimer geometries obtained at the MP2/6-31⫹G* level. As is seen fromTable 3, where the results of these calculations are presented, the increasing of the basis set reduced the BSSE only slightly (by ca 10%) and the difference between the DFT and MP2 BSSE corrections still remains large. This indicates that the difference in the DFT and MP2 BSSE corrections is not because of the difference in the basis set. In the further analysis we have examined whether the larger BSSE error in the MP2 calculations originates from the HF contribution or from the correlation contribution. The comparison of the BSSE’s resulting from the HF/6-31⫹⫹G**//MP2/631⫹⫹G* and the MP2/6-31⫹⫹G**//MP2/631⫹G* calculations for Dimer A equal to 0.001340 and 0.004549 hartree, respectively, indicates that the error is much more significant in the correlation contribution than in the HF contribution. In the matter of fact, the HF error is very similar to the DFT error. Our interpretation of the above results is the

following. It is well known fact, that the DFT method does not include the long-range electron correlation effects, which contribute to the dispersion interactions. These effects are included in the MP2 results. It can be expected that the BSSE is more related to the long-range correlation effects than to the short-range effects as it did results from the presence in the basis set of orbitals located on the other monomer. Therefore, the BSSE in the DFT results should be similar to the error, which one gets in the HF results, and this is what we find in our calculations. However, the BSSE in the MP2 results, which include the long-range correlation effects, should be greater than the HF and DFT BSSE’s, and this is what we also see in our results. In conclusion, the results presented in this work demonstrate that Acr–m 1Ura dimers are planar. In these dimers, l-methyluracil and acrylamide, which models the natural aminoacid Asparagine and Glutamine side chains, interact via the N–H…O H-bonds. We found a good agreement between the experimental enthalpy of formation of 1-methyluracilacrylamide dimer (⫺40.6 ^ 4.2 kJ mol ⫺1) and the interaction energies calculated at the DFT/B3LYP/631⫹⫹G** and MP2/6-31⫹G* levels of theory for the two equilibrium dimer structures. As the two dimers have very similar interaction energies, they could not be distinguished in the present experiment.

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