Effects of Hydrogen-Bonding and Stacking Interactions with Amino

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Effects of Hydrogen-Bonding and Stacking Interactions with Amino Acids on the Acidity of Uracil Ken C. Hunter,† Andrea L. Millen,† and Stacey D. Wetmore*,‡ Department of Chemistry, Mount Allison UniVersity, 63C York Street, SackVille, New Brunswick E4L 1G8, Canada, and Department of Chemistry & Biochemistry, UniVersity of Lethbridge, 4401 UniVersity DriVe, Lethbridge, Alberta T1K 3M4, Canada ReceiVed: October 20, 2006; In Final Form: NoVember 21, 2006

The effects of hydrogen-bonding interactions with amino acids on the (N1) acidity of uracil are evaluated using (B3LYP) density functional theory. Many different binding arrangements of each amino acid to three uracil binding sites are considered. The effects on the uracil acidity are found to significantly depend upon the nature of the amino acid and the binding orientation, but weakly depend on the binding site. Our results reveal that in some instances small models for the amino acids can be used, while for other amino acids larger models are required to properly describe the binding to uracil. The gas-phase acidity of uracil is found to increase by up to approximately 60 kJ mol-1 due to discrete hydrogen-bonding interactions. Although (MP2) stacking interactions with aromatic amino acids decrease the acidity of uracil, unexpected increases in the acidity are found when any of the aromatic amino acids, or the backbone, hydrogen bond to uracil. Consideration of enzymatic and aqueous environments leads to decreases in the effects of the amino acids on the acidity of uracil. However, we find that the magnitude of the decrease varies with the nature of the molecule bound, as well as the (gas-phase) binding orientations and strengths, and therefore solvation effects should be considered on a case-by-case basis in future work. Nevertheless, the effects of amino acid interactions within enzymatic environments are as much as approximately 35 kJ mol-1. The present study has general implications for understanding the nature of active site amino acids in enzymes, such as DNA repair enzymes, that catalyze reactions involving anionic nucleobase intermediates.

Introduction The field of biochemistry revolves around studying the structure, function, and interactions of nucleic acids, which store and transmit genetic information, and proteins, which are biological catalysts. Understanding the interactions between the components of these biomolecules, in particular the nucleobases and amino acids, is especially important because these interactions facilitate key biological functions ranging from protein synthesis to DNA repair. Although recognition of nucleic acids by proteins primarily occurs due to interactions with charged phosphate groups (especially in RNA-protein complexes),1 these interactions are not discriminative, and thus selective recognition of nucleobases by proteins is the result of weaker (hydrogen-bonding and stacking) interactions.2 Because of the importance of nucleobase-amino acid contacts in nature, these interactions have been studied in a variety of different ways. For example, nucleobase-amino acid interactions used by numerous enzymes have been compiled in databases.3-8 These databases are subsequently used to identify relationships between the interactions used by different proteins to recognize specific nucleobase sequences. Alternatively, nucleobase-amino acid interactions have been studied to determine whether amino acids can be used as probes to selectively distinguish between the natural base pairs.9-11 The geometries and binding strengths of some nucleobaseamino acid interactions have been characterized, where theoreti* Corresponding author. E-mail: [email protected]. † Mount Allison University. ‡ University of Lethbridge.

cal (ab initio and density functional) calculations have proven to provide particularly useful information.12-18 Despite these previous computational studies, little has been done to study how these interactions affect the properties of the nucleobase. Specifically, we are interested in the effects of amino acid interactions on the (N1) acidity of uracil. This interest stems from evidence of the formation of a uracil anion upon cleavage of the deoxyuridine glycosidic bond by uracil DNA glycosylase (UDG), a DNA repair enzyme.19,20 Additionally, interactions between active site residues and the substrate (uracil) have been implicated in the catalytic role of this enzyme, where these interactions may directly affect the acidity of uracil.21 Previous work in our laboratory studied the effects of hydrogen-bonding interactions with three small molecules (XH ) HF, H2O, NH3) on the (N1) acidity of uracil,22 as well as the acidity of other nucleobases.23-25 The small molecules were chosen because the proton affinities and acidities span those of the amino acids. Additionally, hydroxyl and amino groups are present in many amino acid side chains, which suggests that at least some amino acids may form hydrogen-bonding patterns similar to these small molecules. Nevertheless, it is important to consider the discrete interactions between amino acids and natural nucleobases. In the present study, we consider the effects of weak (hydrogen-bonding and stacking) interactions with amino acids on the (N1) acidity of uracil. We consider hydrogen-bonding interactions with side chains, where the most notable functional groups include hydroxyl (Ser, Thr, Tyr), thiol (Cys), carboxamide (Asn, Gln), carboxyl (Asp, Glu), amino (Lys), and guanidine (Arg). However, aromatic amino acids (His, Tyr, Trp,

10.1021/jp066902p CCC: $37.00 © 2007 American Chemical Society Published on Web 01/26/2007

Acidity of Uracil

Figure 1. Hydrogen-bonded complexes between uracil and a variety of small molecules (XH ) HF, OH2, NH3, and CH4).

Figure 2. Thermodynamic cycle for the deprotonation of uracil (U) hydrogen bonded to another molecule (XH).

Phe), as well as the peptide backbone, can also form hydrogenbonding interactions with the nucleobase, and therefore the effects of these interactions are investigated. Furthermore, because aromatic amino acids are perhaps best known for their stacking ability, the effects of stacking with these molecules on the acidity of uracil are discussed for completion. From this study, we hope to identify the amino acids that can provide the greatest stabilization to the uracil anion, and thereby enhance our understanding of the nature of enzymatic active site residues that catalyze glycosidic bond cleavage reactions. Computational Details Hydrogen-bonding interactions between three uracil binding sites (Figure 1) and three small molecules (HF, H2O, NH3) were previously studied22 using B3LYP/6-311+G(2d,p) single-point calculations on B3LYP/6-31+G(d,p) optimized geometries. Because our long-term goal is to continuously extend our models in size to adequately depict active site interactions, it would be beneficial to identify a smaller basis set with equal accuracy. Therefore, we compare the acidities of the uracil-XH complexes optimized with the 6-31G(d) and 6-31+G(d,p) basis sets (Table S1, Supporting Information). The binding strengths were also considered because the effect of the small molecule on the acidity (∆(acidity)) is equal to the difference in the binding strengths of the neutral and anionic complexes, as shown by a simple thermodynamic cycle (Figure 2). Specifically, weak binding to neutral uracil or strong binding to the uracil anion will lead to large effects of hydrogen bonding on the uracil acidity. The binding strengths calculated with the 6-31G(d) geometries are generally as much as approximately 5-6 kJ mol-1 weaker than those calculated with the 6-31+G(d,p) geometries, where the decrease in the anionic binding strength is slightly larger than the decrease in the corresponding neutral binding strength for any given small molecule and binding site. The largest differences in binding occur due to variations in the geometries of the anionic complexes (Figures S1-S3, Supporting Information). Specifically, the N3-H‚‚‚X hydrogen-bond distance in the O2(N3) and O4(N3) and the C5-H‚‚‚X distance in the O4(C5) anionic complexes significantly decrease without diffuse functions, where interactions with the nucleobase donor site are not favorable upon anion formation. The differences in geometries and binding strengths lead to weaker acidities when the 6-31G(d) optimized complexes are used (Table S1). However, the decreases in the acidity are

J. Phys. Chem. B, Vol. 111, No. 7, 2007 1859 typically less than 2 kJ mol-1. Furthermore, because the 6-31G(d) geometries decrease the acidity of isolated uracil by only 0.1 kJ mol-1 as compared to 6-31+G(d,p) geometries, there is a very small basis set effect on the effect of hydrogen bonding on the acidity (∆(acidity)). The small differences in the energetics obtained with the two basis sets are somewhat surprising given the significant differences in the distance between the small molecule and the uracil donor in some complexes (Figures S1-S3). The similar energetics for the two sets of geometries may arise due to a flat potential energy surface for the anionic dimers, which has been seen for related cytosine complexes,26 and/or the greater dependence of the stability of the anionic complexes on interactions with the uracil acceptor site, where both basis sets yield similar geometrical parameters for these hydrogen-bonding interactions. Therefore, performing geometry optimizations with 6-31G(d) rather than 6-31+G(d,p) will lead to small changes in the binding strengths, acidity, and effects of hydrogen bonding on the acidity. Based on the above results, uracil-amino acid complexes were optimized, and zero-point vibrational energy corrections obtained, in the gas phase with B3LYP/6-31G(d). Models of most amino acid residues were obtained by replacing the backbone (including the R carbon) with hydrogen. However, additional carbon atoms in the side chain were neglected for some amino acids under the assumption that they do not significantly affect the binding strength. For example, Thr was modeled as Ser (methanol), Gln as Asn (acetamide), and Glu as Asp (formic acid). Although many minima exist for any given uracil-amino acid complex, we limit our investigation to the lowest energy minimum for each unique binding arrangement with each amino acid isomer. Gas-phase single-point energies were computed using B3LYP/6-311+G(2d,p). To assess environmental effects, solvent-phase B3LYP/6311+G(2d,p) single-point calculations were performed on B3LYP/6-31G(d) gas-phase geometries with the PCM method.27 The 6-31G(d) geometries were again used with confidence due to small differences in the results when 6-31+G(d,p) geometries are implemented (see Table S2, Supporting Information). Two dielectric constants were used: 4.335 (ether) and 78.39 (water). Water was chosen due to its abundance in biological systems, while ether ( ) 4.335) was chosen because dielectric constants around  ) 4 have been shown to provide a suitable compromise between the environment within enzymatic active sites and the surrounding water.28 All relative energies include (gas-phase) basis set superposition error (BSSE) corrections.29 Because it is well documented that B3LYP cannot accurately describe stacking interactions, we use MP2/6-31G*(0.25)30 to evaluate the stacking interactions between the aromatic amino acids (His, Trp, Tyr, Phe) and uracil. This method has been shown to provide a reasonable compromise between accuracy and cost when used to study the stacking interactions between DNA nucleobases.31-35 Because full optimizations of stacked systems typically lead to distorted geometries and/or hydrogenbonded structures,36 we use potential energy surface scans to determine the best stacked geometry. Uncomplexed uracil, the uracil anion, and the aromatic amino acids, where the methyl groups of the side chains were removed, were initially optimized with MP2/6-31G(d) in Cs symmetry. The monomers were stacked by aligning the centers of mass and overlapping the model glycosidic and backbone bonds (Figure S4, Supporting Information). Subsequently, the optimum vertical displacement was determined (by varying the distance by 0.1 Å). Starting from the structure with the optimum vertical displacement, the preferred angle of rotation was identified by rotating the amino

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TABLE 1: Calculated Binding Strengths of Neutral and Anionic Uracil-XH Complexes, the (N1) Acidity of Uracil-XH Complexes, and the Effects of Hydrogen Bonding with XH on the (N1) Acidity of Uracil (∆(acidity)), Where XH ) HF, H2O, NH3, and CH4 (kJ mol-1)a O2(N3)

O4(N3)

O4(C5)

HF HF HF H2O H2O H2O NH3 NH3 NH3 CH4 CH4 CH4

neutral BS 32.2 36.8 34.1 18.7 21.2 14.7 21.3 22.3 9.3 -2.4 -2.4 -2.2

anion BS

acidity

∆(acidity)

82.1 77.6 79.0 37.2 34.9 37.6 15.0 13.5 18.2 1.4 1.4 1.8

1389.5 1339.6 1348.7 1344.6 1371.0 1375.8 1366.6 1395.8 1398.3 1380.6 1385.7 1385.7 1385.5

49.9 40.8 44.9 18.5 13.7 22.9 -6.3 -8.8 8.9 3.8 3.8 4.0

a

Binding strengths and acidities were calculated at the B3LYP/6-311+G(2d,p)//B3LYP/6-31G(d) level and include BSSE and ZPVE corrections. See Figure 1 for depiction of uracil binding sites and Figure 3 for representative binding geometries.

acid counter clockwise in 30° increments from 0 to 360°. All stacking energies include BSSE corrections.29 All calculations were performed with Gaussian 03.37 Results and Discussion Complexes with Small Molecules (HF, H2O, NH3, CH4) in the Gas Phase. As mentioned in the Introduction, studies of the effects of hydrogen-bonding interactions with small molecules are an important first step to understanding interactions with amino acids. Therefore, our previous work bound three small molecules (XH ) HF, H2O, NH3) to three uracil binding sites (Figure 1). In the present study, we extend upon our previous small models by considering uracil complexes with XH ) CH4 (Table 1). Uracil-methane complexes were considered because many amino acid side chains are aliphatic. Additionally, C-H hydrogen bonds have been shown to have significant binding strengths38-53 and have been suggested to play important roles in many biological processes.54-59 The geometries for the uracil-XH O2(N3) complexes are displayed in Figure 3, which provide representative illustrations of the interesting geometrical features in all uracil complexes. Our calculations show that methane binds to uracil through one C-H‚‚‚O hydrogen bond and the methyl group is eclipsed with respect to the uracil donor in the O2(N3) and O4(N3) complexes and staggered in the O4(C5) complex. Hydrogen fluoride also forms one (strong) hydrogen bond with uracil, while ammonia and water act as both a proton donor and acceptor and therefore form bidendate hydrogen bonds with uracil. Upon anion formation, the small molecules move closer to the uracil acceptor and further from the uracil donor, while remaining in the molecular plane of the complex. The non-hydrogen-bonding water hydrogen is located out of the uracil molecular plane in both the neutral and the anionic complexes. The data in Table 1 reveal that neutral uracil binds the strongest to HF. Although H2O and NH3 bind weaker to uracil than HF, the corresponding water and ammonia complexes have similar binding strengths due to the fact that these small molecules form bidentate binding arrangements with uracil, and therefore the total binding strength is dependent on a balance between the strength of interactions with the uracil donor and acceptor. Despite previously reported positive binding strengths for the methane-formaldehyde dimer calculated at a comparable level of theory,41 the binding strengths of the neutral uracilmethane complexes are slightly negative. However, the previous work on the methane-formaldehyde dimer fixed the hydrogenbond angle,41 and it is therefore not clear whether zero-point

Figure 3. Selected B3LYP/6-31G(d) bond lengths (Å) and angles (deg) in (a) neutral and (b) anionic O2(N3) uracil-XH complexes, where XH ) HF, H2O, NH3, and CH4.

corrections were included. Indeed, we note that neglecting zeropoint corrections leads to positive binding strengths for methaneuracil complexes with magnitudes (0.5-0.6 kJ mol-1) comparable to those reported for the methane-formaldehyde dimer, where this discrepancy likely arises due to the low-frequency vibrational modes in these weak complexes. The anionic binding strengths decrease as HF > H2O > NH3 > CH4 at all binding sites. The methane binding strengths to the uracil anion are positive, where the binding strengths further increase (to 4.8-5.4 kJ mol-1) upon removal of zero-point

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J. Phys. Chem. B, Vol. 111, No. 7, 2007 1861

Figure 4. Binding orientations, as well as selected B3LYP/6-31G(d) bond lengths (Å) and angles (deg), in neutral uracil O2(N3) complexes with Ser (Thr), Tyr, and Cys.

corrections. The ammonia anionic complexes show weaker binding than the corresponding water complexes due to the weak acidity of N-H bonds and unfavorable interactions between the uracil (anion) donor and the (basic) ammonia acceptor. The effect of hydrogen bonding on the acidity of uracil (∆(acidity)) generally decreases with the acidity of the small molecule bound (Table 1). Specifically, the effect on the acidity decreases as HF > H2O > CH4 > NH3 for binding at O2(N3) and O4(N3), and as HF > H2O > NH3 > CH4 at O4(C5). Interestingly, ammonia decreases the acidity at two binding sites by up to 9 kJ mol-1, while methane increases the acidity at all binding sites by approximately 4 kJ mol-1. We note that the effects of methane on the acidity calculated using the (positive) binding strengths obtained without zero-point corrections fall in the range of 4.3-4.8 kJ mol-1, and therefore we are confident in the magnitude of the effects of methane on the acidity. The effect of hydrogen bonding on the acidity is dependent on the binding site for all small molecules. Specifically, for most small molecules, the effect decreases as O4(C5) > O2(N3) > O4(N3) with the exception of XH ) HF, where the effect decreases as O2(N3) > O4(C5) > O4(N3). The trend with respect to the uracil binding site occurs due to a balance between the proton affinity and acidity of the uracil binding sites as discussed previously in the literature.22,23 Nevertheless, ∆(acidity) is more dependent on the nature of the small molecule bound than the binding site. In summary, differences in the binding strengths of neutral and anionic uracil complexes lead to significant effects of hydrogen bonding with small molecules on the acidity of uracil, which are more dependent on the nature of the small molecule bound than the binding site. The results from this small molecule study will provide a foundation for discussing and understanding the effects of interactions with the natural amino acids on the acidity of uracil. Complexes with Amino Acids in the Gas Phase. As mentioned in the Computational Details, we will investigate the effects of interactions with the amino acids on the acidity of uracil in the gas and solvent (ether and water) phases. However, to simplify our discussion, we will first consider the geometries, binding strengths, and acidities of the gas-phase complexes. Subsequently, the environmental effects will be discussed. As will be seen, the effect on the acidity of uracil strongly depends on the nature of the amino acid, but weakly depends on the binding site, which is the same trend discussed for the small molecules. Therefore, we primarily focus our discussion on the magnitude of the effects in relation to the identity and binding orientation of the amino acid. (i) Hydroxyl or Thiol Group. Ser and Thr (methanol) bind to uracil in one orientation, where the hydroxyl group acts as both a proton acceptor and donor, and the methyl group is located out of the uracil molecular plane (Figure 4). Because of similarities in the binding orientations, Ser (Thr) complexes have only slightly stronger binding strengths (Table 2) than the

corresponding water complexes (Table 1), where the binding strengths for the neutral Ser (Thr) O2(N3) and O4(N3) complexes have been previously reported in the literature.8,60 Because of the small differences in binding, Ser (Thr) has only a slightly greater (by up to 2.3 kJ mol-1) effect on the acidity of uracil as compared to water. This supports our assumption that additional carbon atoms in the side chain lead to small changes in the nucleobase-amino acid interactions (i.e., Ser and H2O yield similar results, and therefore Thr can be modeled as methanol). Tyr (4-methylphenol) complexes to uracil in an orientation similar to that of Ser(Thr), where the hydroxyl group acts as both a proton donor and acceptor, and the phenyl group is located out of the uracil molecular plane (Figure 4). However, we find that Tyr binds slightly stronger than Ser (Thr) to neutral uracil, and significantly stronger to the uracil anion (Table 2). The end result is that Tyr increases the acidity of uracil by roughly 20 kJ mol-1 more than Ser (Thr). This increase arises because Tyr is more acidic than Ser (Thr) due to stabilization provided by the phenyl group. Indeed, Tyr behaves closer to HF than H2O. These findings contradict suggestions from Cheng et al.,8 who hypothesized that Tyr interactions can be accurately represented as Ser (Thr) despite the extra bulk of the Tyr ring. The thiol group of cysteine (Cys) leads to very small changes in the binding orientation as compared to the hydroxyl group of Ser (Thr) (Figure 4). However, replacing oxygen with sulfur leads to longer hydrogen bonds, and thus weakens the binding in both the neutral (by up to 13.8 kJ mol-1) and the anionic (by up to 19.0 kJ mol-1) complexes (Table 2). These differences in binding increase the acidity of uracil by 10-19 kJ mol-1. Thus, the effects of interactions with Cys on the acidity of uracil fall only slightly (approximately 4 kJ mol-1) below those reported for water. (ii) Carboxamide Group. Asn and Gln (acetamide) complex to uracil in two different orientations (denoted as Asn(Gln)-1 and Asn(Gln)-2, Figure 5). The amino group bonding arrangement in Asn(Gln)-1 is similar to that in the corresponding ammonia complex (Figure 3). However, the methyl group of Asn also comes within hydrogen-bonding distance to the uracil carbonyl (Figure 6), where the contact distances are similar to those seen in the uracil-methane complexes (Figure 3). Contrary to the comparison between Ser (Thr) and H2O, Asn (Gln) produces significantly different effects on the acidity as compared to NH3. Specifically, the acidity of the Asn(Gln)-1 complex is 42.4-46.7 kJ mol-1 (Table 3) larger than the acidity of uracil, and therefore the effects of Asn are more similar to HF than NH3 (Table 1). These differences arise due to differences in the neutral and anionic binding strengths. Most importantly, the binding strengths of the Asn(Gln)-1 anionic complexes are up to 40.6 kJ mol-1 larger than the corresponding ammonia complexes. Stronger binding arises for Asn due to resonance within the amino acid, which places a net positive charge on the amino group and a net negative charge on the

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TABLE 2: Binding Strengths of Neutral and Anionic Complexes, the N1 Acidity of Complexes, the Effects of Solvation on the Acidity (∆(solv)), and the Effects of Hydrogen Bonding on the Nucleobase Acidity (∆(acidity)) When Ser, Tyr, or Cys Is Bound to Uracil in a Variety of Environments (kJ mol-1)a gasb

Tyr

Cys

OH

OH

SH

waterc

neutral BS anion BS acidity ∆(acidity)d acidity ∆(solv)e ∆(acidity)d acidity ∆(solv)e ∆(acidity)d

acceptor donor Ser(Thr)

etherc

OH O2(N3) O4(N3) O4(C5)

22.9 25.5 17.5

42.9 40.3 42.7

1369.5 1374.7 1364.3

20.0 14.8 25.2

1241.7 1245.1 1241.5

127.8 129.6 122.8

10.4 7.0 10.6

1206.3 1208.8 1207.6

163.2 165.9 156.7

8.8 6.3 7.5

O2(N3) O4(N3) O4(C5)

24.0 27.1 23.8

68.9 65.4 69.3

1344.6 1351.2 1344.0

44.9 38.3 45.5

1231.6 1236.2 1235.6

113.0 115.0 108.4

20.5 15.9 16.5

1198.3 1201.2 1205.1

146.3 150.0 138.9

16.8 13.9 10.0

O2(N3) O4(N3) O4(C5)

9.1 12.0 6.1

23.9 22.5 24.7

1374.7 1379.0 1370.9

14.8 10.5 18.5

1249.8 1256.9 1250.6

124.9 122.1 120.3

2.3 -4.8 1.5

1213.9 1221.9 1217.2

160.8 157.1 153.7

1.2 -6.8 -2.1

OH

SH

a See Figure 1 for depiction of uracil binding sites and Figure 4 for amino acid binding orientations. b Gas-phase B3LYP/6-311+G(2d,p) singlepoint calculations were performed on gas-phase B3LYP/6-31G(d) geometries. c PCM-B3LYP/6-311+G(2d,p) single-point calculations were performed on gas-phase B3LYP/6-31G(d) geometries. d The difference between the acidity of the complex and the acidity of isolated uracil in the same phase. e The difference between the acidity of the complex in solution and in the gas phase.

Figure 5. Binding orientations, as well as selected B3LYP/6-31G(d) bond lengths (Å) and angles (deg), in neutral uracil O2(N3) complexes with Asn (Gln).

Figure 6. Comparison of selected B3LYP/6-31G(d) bond lengths (Å) and angles (deg) in the Asn(Gln)-1 (a) neutral and (b) anionic uracil O2(N3) complexes.

carbonyl. The additional interaction with the methyl group also likely plays a role as we previously saw that methane can increase the acidity of uracil by up to 4 kJ mol-1 (Table 1). Our calculated binding strengths are smaller than those previously reported.15-17 Nevertheless, we find that the neutral Asn(Gln)-2 complexes are as much as 28.1 kJ mol-1 stronger than the neutral Asn(Gln)-1 complexes due to the utilization of different amino acid donor and acceptor sites. However, Asn(Gln)-2 has weaker anionic complexes (by up to 21.6 kJ mol-1), and therefore Asn in this binding arrangement has a smaller effect on the nucleobase acidity. Indeed, the O4(N3) Asn(Gln)-2 complex has a smaller acidity than uracil (by -6.6 kJ mol-1), while the other two complexes have slightly larger (by up to 9.9 kJ mol-1) acidities. In summary, Asn (Gln) can have a significant effect on the acidity of uracil. However, this effect is largely dependent on the binding configuration, and thus the binding orientation within the active sites of enzymes must be carefully assessed. Indeed, Asn binds to uracil (at O4(N3)) within the active site of uracil DNA glycosylase in an arrangement most similar to Asn(Gln)-2 and, therefore, may not play an important role in base anion stabilization.

(iii) Carboxyl Group. Although carboxyl groups may appear in unprotonated (anionic) forms within the active sites of enzymes, these residues would have unfavorable interactions with the uracil anion. Thus, we use neutral acetic acid models in the present study to allow comparisons with the other amino acid functional groups. It should be noted that Asp and Glu have been previously modeled as (neutral) formic acid.16 The hydroxyl group in Asp (Glu) can adopt a cis or trans orientation with respect to the carbonyl,61 and the two isomers lead to three distinct uracil complexes (Figure 7). Asp(Glu)-1, which uses the cis isomer to coordinate to uracil solely through the hydroxyl group, has a binding arrangement similar to those discussed for water, Ser (Thr), and Tyr. Indeed, in this configuration, Asp (Glu) binds to uracil (Table 4) 3 kJ mol-1 weaker than Ser (Thr) (Table 2) at O2(N3) and O4(N3), but binds 3 kJ mol-1 stronger at O4(C5). However, the anionic binding strengths are approximately 22 kJ mol-1 stronger for Asp (Glu) because the neighboring carbonyl increases the acidity of the hydroxyl group. Indeed, the O2‚‚‚H distance is 1.491 Å, which indicates that a proton is partially transferred to the uracil anion. This leads to significant (up to 45 kJ mol-1) increases in the acidity of uracil upon binding Asp (Glu), which is similar to the increase provided by interactions with Tyr or HF. The Asp(Glu)-2 complex (Figure 7) has a uracil binding arrangement similar to that of Asp(Glu)-1, but involves the higher-energy, and therefore more acidic, trans isomer. Thus, the neutral binding strengths for Asp(Glu)-2 are stronger (by 7.8-12.1 kJ mol-1) as compared to Asp(Glu)-1, while the anionic binding strengths are significantly stronger (by 22.624.6 kJ mol-1). A portion of the increased binding may also be due to interactions between the amino acid methyl group and the uracil carbonyl, which were previously discussed for Asn(Gln)-1. These differences in binding lead to large increases (by up to 61 kJ mol-1) in the acidity of uracil upon binding of the trans isomer, which is much larger than the increase discussed for the cis isomer or Tyr. The final Asp (Glu) complex (Asp(Glu)-3, Figure 7) has been previously reported in the literature.16 As discussed for Asn (Gln), using a different donor and acceptor site strengthens the binding to (neutral) uracil by up to approximately 25 kJ mol-1 as compared to Asp (Glu) binding orientations. On the other hand, the anionic binding increases by up to 9 kJ mol-1 as compared to Asp(Glu)-1 and decreases as compared to Asp(Glu)-2 by up to 24 kJ mol-1. Thus, the large increase in the

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TABLE 3: Binding Strengths of Neutral and Anionic Complexes, the N1 Acidity of Complexes, the Effects of Solvation on the Acidity (∆(solv)), and the Effects of Hydrogen Bonding on the Nucleobase Acidity (∆(acidity)) When Asn (Gln) Is Bound to Uracil in a Variety of Environments (kJ mol-1)a gasb Asn(Gln)-1

Asn(Gln)-2

waterc

neutral BS anion BS acidity ∆(acidity)d acidity ∆(solv)e ∆(acidity)d acidity ∆(solv)e ∆(acidity)d

acceptor

donor

NH2

NH2, CH3

OdC

etherc

O2(N3) O4(N3) O4(C5)

11.3 13.5 14.8

58.0 55.9 59.9

1342.8 1347.1 1344.4

46.7 42.4 45.1

112.7 115.0 106.8

22.0 20.0 14.5

112.7 115.0 106.8

145.6 150.0 136.1

145.6 150.0 136.1

17.9 18.0 6.8

O2(N3) O4(N3) O4(C5)

38.1 41.6 28.4

39.2 35.1 38.3

1388.4 1396.0 1379.6

1.1 -6.5 9.9

134.4 137.1 128.7

-1.9 -6.8 1.2

134.4 137.1 128.7

173.0 177.4 165.9

173.0 177.4 165.9

-0.3 -3.5 1.4

NH2

a See Figure 1 for depiction of uracil binding sites and Figure 5 for amino acid binding orientations. b Gas-phase B3LYP/6-311+G(2d,p) singlepoint calculations were performed on gas-phase B3LYP/6-31G(d) geometries. c PCM-B3LYP/6-311+G(2d,p) single-point calculations were performed on gas-phase B3LYP/6-31G(d) geometries. d The difference between the acidity of the complex and the acidity of isolated uracil in the same phase. e The difference between the acidity of the complex in solution and in the gas phase.

Figure 7. Binding orientations, as well as selected B3LYP/6-31G(d) bond lengths (Å) and angles (deg), in neutral uracil O2(N3) complexes with Asp (Glu).

TABLE 4: Binding Strengths of Neutral and Anionic Complexes, the N1 Acidity of Complexes, the Effects of Solvation on the Acidity (∆(solv)), and the Effects of Hydrogen Bonding on the Nucleobase Acidity (∆(acidity)) When Asp (Glu) Is Bound to Uracil in a Variety of Environments (kJ mol-1)a gasb acceptor Asp(Glu)-1

Asp(Glu)-2

Asp(Glu)-3

OH

OH

donor OH

etherc

waterc

neutral BS anion BS acidity ∆(acidity)d acidity ∆(solv)e ∆(acidity)d acidity ∆(solv)e ∆(acidity)d

isomer cis O2(N3) O4(N3) O4(C5)

20.1 22.0 20.9

64.6 61.8 64.3

1345.0 1349.7 1346.1

44.5 39.8 43.3

1226.4 118.6 1229.8 119.9 1232.3 113.8

25.7 22.3 19.8

1195.3 149.7 1196.1 153.6 1201.5 144.6

19.8 19.0 13.6

O2(N3) O4(N3) O4(C5)

27.9 34.1 32.1

88.6 84.4 88.9

1328.8 1339.2 1332.7

60.7 50.3 56.8

1219.1 109.7 1225.6 113.6 1227.8 104.9

33.0 26.5 24.3

1189.1 139.7 1193.4 145.8 1200.1 132.6

26.0 21.7 15.0

O2(N3) O4(N3) O4(C5)

44.1 48.2 35.0

73.5 68.0 65.0

1360.1 1369.7 1359.5

29.4 19.8 30.0

1234.0 126.1 1240.9 128.8 1238.2 121.3

18.1 11.2 13.9

1199.3 160.8 1204.9 164.8 1204.4 155.1

15.8 10.2 10.7

OH, CH3 trans

OdC OH

cis

a See Figure 1 for depiction of uracil binding sites and Figure 7 for amino acid binding orientations. b Gas-phase B3LYP/6-311+G(2d,p) singlepoint calculations were performed on gas-phase B3LYP/6-31G(d) geometries. c PCM-B3LYP/6-311+G(2d,p) single-point calculations were performed on gas-phase B3LYP/6-31G(d) geometries. d The difference between the acidity of the complex and the acidity of isolated uracil in the same phase. e The difference between the acidity of the complex in solution and in the gas phase.

neutral binding strength reduces the overall effect of Asp (Glu) on the acidity by 13.3-31.3 kJ mol-1 as compared to other Asp (Glu) binding orientations. Nevertheless, the acidity of the Asp(Glu)-3 complex is 19.8-30.0 kJ mol-1 greater than the acidity of uracil, which is a larger effect than seen for water or Ser (Thr). In summary, neutral Asp (Glu) has significant effects on the acidity of uracil, where the magnitude of the effect is largely dependent on the isomer bound and the binding orientation. We note, however, that the amino acid is likely deprotonated within enzymatic environments, and therefore close contacts would instead destabilize the uracil anion. (iV) Amino Group. Like ammonia, Lys (butan-1-amine) coordinates to uracil solely through the amino group (Figure 8). Although a similar binding arrangement was found for Asn (Gln), the binding strengths of the uracil-Lys complexes (Table 5) are closer to those of the uracil-NH3 complexes (Table 1).

Indeed, Lys and ammonia lead to similar effects on the acidity of uracil. Thus, adding trans-butane to the amino group has a small effect on the binding strengths and the effects of hydrogen bonding on the acidity, which further validates our truncated models for other amino acids. (V) Guanidine Group. For consistency with the other amino acids discussed thus far, we consider the neutral form of Arg. The most complete model of the (neutral) Arg side chain is propylguanidine. However, due to the flexibility of the carbon chain, the number of minima and possible binding arrangements may distract from a meaningful comparison to other amino acids. Because we have already shown that additional carbons have small effects on the acidity of uracil complexes, we will consider a shortened Arg chain. Previous studies have used a range of Arg models,8,16,62,63 and, therefore, to determine the most appropriate model, we consider a series of uracil O2(N3)

1864 J. Phys. Chem. B, Vol. 111, No. 7, 2007

Hunter et al.

TABLE 5: Binding Strengths of Neutral and Anionic Complexes, the N1 Acidity of Complexes, the Effects of Solvation on the Acidity (∆(solv)), and the Effects of Hydrogen Bonding on the Nucleobase Acidity (∆(acidity)) When Lys Is Bound to Uracil in a Variety of Environments (kJ mol-1)a gasb NH2

waterc

neutral BS anion BS acidity ∆(acidity)d acidity ∆(solv)e ∆(acidity)d acidity ∆(solv)e ∆(acidity)d

acceptor donor Lys

etherc

NH2 O2(N3) O4(N3) O4(C5)

24.4 25.1 10.0

18.6 16.7 22.7

1395.3 1397.9 1376.8

-5.8 -8.4 12.7

1265.9 1266.6 1253.2

129.4 131.3 123.6

-13.8 -14.5 -1.1

1228.1 1227.6 1215.5

167.2 170.3 161.3

-13.0 -12.5 -0.4

a See Figure 1 for depiction of uracil binding sites and Figure 8 for amino acid binding orientations. b Gas-phase B3LYP/6-311+G(2d,p) singlepoint calculations were performed on gas-phase B3LYP/6-31G(d) geometries. c PCM-B3LYP/6-311+G(2d,p) single-point calculations were performed on gas-phase B3LYP/6-31G(d) geometries. d The difference between the acidity of the complex and the acidity of isolated uracil in the same phase. e The difference between the acidity of the complex in solution and in the gas phase.

Figure 8. Binding orientation, as well as selected B3LYP/6-31G(d) bond lengths (Å) and angles (deg), in neutral uracil O2(N3) complex with Lys.

complexes with Arg models containing chains of zero to two trailing carbons in length. When Arg is modeled as guanidine, five unique binding orientations to uracil can be found that differ in the nature of the amino acid donor and acceptor (Arg-H-1-Arg-H-5, Figure 9). Expansion of the Arg model to methylguanidine or ethylguanidine allows differentiation between the amino groups in guanidine, where one becomes a secondary amine. Furthermore, methylguanidine and ethylguanidine can adopt four important isomers (A-D, Figure 10).64 Therefore, the total number of possible binding configurations increases, where 10 unique binding arrangements for both models can be found (Arg-CH2R-1-Arg-CH2R-10, Figure 9). Table S3 (Supporting Information) compares the O2(N3) binding orientations and provides the corresponding binding strengths for each Arg model. Based on the increase in the number of binding arrangements alone, the guanidine model does not accurately depict the possible binding orientations of Arg. However, the binding orientations and corresponding energetics for both the methylguanidine and the ethylguanidine models are very similar, where, for example, the acidities are within 1.3 kJ mol-1. Therefore, although methylguanidine is likely a suitable Arg model, we use ethylguanidine to consider binding at all three uracil sites with confidence that expansion to propylguanidine will produce similar results. Table 6 contains the binding strengths of the uracil-Arg complexes, as well as the effects of Arg on the acidity of the nucleobase. Differences in the interaction energies as compared to ammonia are found for most binding arrangements because resonance within the guanidine group places a net negative charge on the imino group and a net positive charge on the (terminal) amino group, which makes this group more acidic than ammonia. Furthermore, although the magnitude of the binding strengths for any given binding orientation is not strongly dependent on the isomer bound, there are significant differences in the binding strengths with respect to the binding orientation, which arises due to differences in the acid-base properties of amino acid donor and acceptor sites. Differences in the binding strengths also arise due to significant geometrical differences among the binding orienta-

Figure 9. Different binding orientations of guanidine (Arg-H), methylguanidine (Arg-CH2R, R ) H), and ethylguanidine (Arg-CH2R, R ) CH3) Arg models to uracil, where arrows identify amino acids acceptor and donor sites.

Figure 10. Nomenclature of four isomers of methylguanidine (R ) H) and ethylguanidine (R ) CH3).

tions. For example, consider the binding modes of Arg-CH2R1, Arg-CH2R-2, and Arg-CH2R-6, which all use the terminal amino group as both a donor and acceptor (Figure 11) ArgCH2R-1 has a binding arrangement very similar to that of Asn(Gln)-1 (Figure 6), which involves a C-H donor. Arg-CH2R2, on the other hand, has stronger binding than Arg-CH2R-1

Acidity of Uracil

J. Phys. Chem. B, Vol. 111, No. 7, 2007 1865

TABLE 6: Binding Strengths of Neutral and Anionic Complexes, the N1 Acidity of Complexes, the Effects of Solvation on the Acidity (∆(solv)), and the Effects of Hydrogen Bonding on the Nucleobase Acidity (∆(acidity)) When Arg (Arg-CH2R, R ) CH3) Is Bound to Uracil in a Variety of Environments (kJ mol-1)a gasb acceptor Arg-CH2R-1 NH2

donor NH2, CH3

B Arg-CH2R-2 NH2

NH2, 2°-NH

C D

Arg-CH2R-3 2°-NH NH2

A B

Arg-CH2R-4 2°-NH NH2, 2°-NH

C D

Arg-CH2R-5 2°-NH NH

C

Arg-CH2R-6 NH2

A

NH2, NH

D Arg-CH2R-7 NH

2°-NH, CH3

A

Arg-CH2R-8 NH

2°-NH, NH

B

Arg-CH2R-9 NH

NH2

B C

Arg-CH2R-10 NH

NH2, NH

A D

waterc

neutral anion BS BS acidity ∆(acidity)d acidity ∆(solv)e ∆(acidity)d acidity ∆(solv)e ∆(acidity)d

isomer A

etherc

O2(N3) O4(N3) O4(C5) O2(N3) O4(N3) O4(C5) O2(N3) O4(N3) O4(C5) O2(N3) O4(N3) O4(C5) O2(N3) O4(N3) O4(C5) O2(N3) O4(N3) O4(C5) O2(N3) O4(N3) O4(C5) O2(N3) O4(N3) O4(C5) O2(N3) O4(N3) O4(C5) O2(N3) O4(N3) O4(C5) O2(N3) O4(N3) O4(C5) O2(N3) O4(N3) O4(C5) O2(N3) O4(N3) O4(C5) O2(N3) O4(N3) O4(C5) O2(N3) O4(N3) O4(C5) O2(N3) O4(N3) O4(C5) O2(N3) O4(N3) O4(C5)

15.1 17.0 12.6 14.7 16.8 12.3 27.8 30.4 15.9 25.6 27.5 15.8 25.4 28.2 18.0 23.1 25.6 15.4 22.3 23.7 13.3 23.5 25.6 16.7 4.2 4.7 15.9 17.1 18.4 14.5 18.1 19.4 14.1 48.4 51.3 23.6 27.0 29.5 18.8 49.4 51.9 25.4 47.0 49.4 23.7 24.2 26.6 17.6 22.7 24.7 15.7

49.5 46.8 50.6 44.2 41.9 48.8 56.3 53.1 57.6 58.5 53.3 59.3 47.3 45.1 49.8 44.7 43.2 46.6 47.6 46.2 57.2 49.1 46.4 58.4 2.7 1.9 16.0 43.4 41.8 44.5 41.6 40.1 43.2 30.0 26.2 21.8 41.7 38.4 43.9 27.7 25.3 21.6 26.0 23.1 19.9 39.2 37.6 43.9 37.5 35.8 42.5

1355.1 1359.7 1351.5 1360.0 1364.4 1353.0 1361.0 1366.8 1347.8 1356.6 1363.7 1346.0 1367.6 1372.6 1357.7 1367.9 1371.9 1358.3 1364.2 1367.0 1345.6 1363.9 1368.7 1347.8 1391.0 1392.3 1389.4 1363.2 1366.1 1359.5 1366.0 1368.8 1360.4 1407.9 1414.6 1391.3 1374.8 1380.6 1364.4 1411.2 1416.1 1393.2 1410.5 1415.8 1393.3 1374.5 1378.5 1363.2 1374.7 1378.4 1362.7

34.4 29.8 38.0 29.5 25.1 36.5 28.5 22.7 41.7 32.9 25.8 43.5 21.9 16.8 31.8 21.6 17.6 31.2 25.3 22.5 43.9 25.6 20.7 41.7 -1.5 -2.8 0.1 26.3 23.4 30.0 23.5 20.7 29.1 -18.4 -25.1 -1.8 14.7 8.9 25.1 -21.7 -26.6 -3.7 -21.0 -26.3 -3.8 15.0 11.0 26.2 14.8 11.1 26.7

1244.0 1246.3 1244.3 1245.1 1247.6 1242.4 1250.1 1252.7 1242.3 1248.8 1253.5 1237.5 1248.8 1252.2 1244.7 1250.3 1253.4 1245.4 1246.9 1249.4 1241.6 1246.8 1250.0 1244.5 1265.8 1266.2 1263.4 1244.6 1246.4 1246.6 1246.1 1248.0 1246.0 1254.3 1256.5 1249.3 1274.4 1277.0 1264.3 1270.1 1273.3 1259.2 1267.7 1270.6 1258.3 1252.9 1255.0 1246.8 1253.4 1255.2 1245.6

111.1 113.4 107.2 114.9 116.8 110.6 110.9 114.1 105.5 107.8 110.2 108.5 118.8 120.4 113.0 117.6 118.5 112.9 117.3 117.6 104.0 117.1 118.7 103.3 125.2 126.1 126.0 118.6 119.7 112.9 119.9 120.8 114.4 153.6 158.1 142.0 100.4 103.6 100.1 141.1 142.8 134.0 142.8 145.2 135.0 121.6 123.5 116.4 121.3 123.2 117.1

8.1 5.8 7.8 7.0 4.5 9.7 2.0 -0.6 9.8 3.3 -1.4 14.6 3.3 -0.1 7.4 1.8 -1.3 6.7 5.2 2.7 10.5 5.3 2.1 7.6 -13.7 -14.1 -11.3 7.5 5.7 5.5 6.0 4.1 6.1 -2.2 -4.4 2.8 -22.3 -24.9 -12.2 -18.0 -21.2 -7.1 -15.6 -18.5 -6.2 -0.8 -2.9 5.3 -1.3 -3.1 6.5

1211.3 1212.3 1214.7 1211.5 1212.5 1209.2 1217.0 1218.0 1211.6 1216.2 1221.6 1207.9 1214.5 1218.3 1212.4 1215.0 1219.5 1213.3 1212.4 1215.4 1215.3 1212.3 1215.3 1218.0 1227.7 1228.8 1223.3 1208.2 1209.0 1213.2 1211.6 1212.0 1213.0 1219.3 1220.8 1216.3 1230.4 1232.6 1223.1 1227.1 1229.6 1218.9 1225.2 1227.7 1217.9 1218.9 1218.5 1213.2 1219.3 1219.3 1212.4

143.8 147.4 136.8 148.5 151.9 143.8 144.0 148.8 136.2 140.4 142.1 138.1 153.1 154.3 145.3 152.9 152.4 145.0 151.8 151.6 130.3 151.6 153.4 129.8 163.3 163.5 166.1 155.0 157.1 146.3 154.4 156.8 147.4 188.6 193.8 175.0 144.4 148.0 141.3 184.1 186.5 174.3 185.3 188.1 175.4 155.6 160.0 150.0 155.4 159.1 150.3

3.8 2.8 0.4 3.6 2.6 5.9 -1.9 -2.9 3.5 -1.1 -6.5 7.2 0.6 -3.2 2.7 0.1 -4.4 1.8 2.7 -0.3 -0.2 2.8 -0.2 -2.9 -12.6 -13.7 -8.2 6.9 6.1 1.9 3.5 3.1 2.1 -4.2 -5.7 -1.2 -15.3 -17.5 -8.0 -12.0 -14.5 -3.8 -10.1 -12.6 -2.8 -3.8 -3.4 1.9 -4.2 -4.2 2.7

a See Figure 1 for depiction of uracil binding sites, Figure 9 for definition of amino acid binding orientations, and Figure 10 for depiction of isomers. b Gas-phase B3LYP/6-311+G(2d,p) single-point calculations were performed on gas-phase B3LYP/6-31G(d) geometries. c PCM-B3LYP/ 6-311+G(2d,p) single-point calculations were performed on gas-phase B3LYP/6-31G(d) geometries. d The difference between the acidity of the complex and the acidity of isolated uracil in the same phase. e The difference between the acidity of the complex in solution and in the gas phase.

in the neutral complex because different sites act as the amino acid donor and acceptor, and in the anionic complexes because anion formation leads to a strongly bound, bridged orientation about the uracil carbonyl. Indeed, the Arg-CH2R-2 complexes have the largest anionic binding strengths among Arg complexes. Arg-CH2R-6 is different yet again from Arg-CH2R-1 and Arg-CH2R-2 because this binding orientation primarily uses the amino group to coordinate to uracil, and the corresponding hydrogen-bond network is located within the uracil molecular plane.

As another example of geometrical differences between binding arrangements, consider Arg-CH2R-7 and Arg-CH2R-8 (Figure 12). Arg-CH2R-7 involves a planar hydrogen-bond network and a large binding strength to neutral uracil. Indeed, along with Arg-CH2R-9, which involves a similar (planar) binding arrangement, Arg-CH2R-7 leads to the strongest binding strengths among neutral uracil-Arg complexes. However, the corresponding anionic complexes also have planar hydrogenbonding orientations, which leads to weaker anionic binding strengths due to unfavorable interactions between the amino acid

1866 J. Phys. Chem. B, Vol. 111, No. 7, 2007

Hunter et al. TABLE 7: Binding Strengths of Neutral and Anionic Complexes, the N1 Acidity of Complexes, and the Effects of Binding on the Nucleobase Acidity (∆(acidity)) When an Aromatic Amino Acid Is Stacked with Uracil (kJ mol-1)a amino acid

neutral BS

anionic BS

acidity

∆(acidity)

Phe Tyr Tyr(f) Trp Trp(f) His His(f)

18.7 25.2 24.4 30.6 30.9 23.1 24.7

7.8 11.2 14.2 11.1 11.8 12.4 11.5

1444.4 1455.3 1458.4 1454.6 1464.0 1463.6 1455.1 1457.7

-10.8 -14.0 -10.2 -19.6 -19.2 -10.7 -13.3

a Binding strengths and acidities were calculated at the MP2/631G*(0.25) level of theory and include BSSE corrections. Stacked geometries were obtained from potential energy surface scans where the preferred vertical displacement and angle of rotation are provided in Table S4 (Supporting Information).

Figure 11. Comparison of binding orientations, as well as selected B3LYP/6-31G(d) bond lengths (Å) and angles (deg), in (a) neutral and (b) anionic uracil O2(N3) complexes with Arg-CH2R-1, Arg-CH2R2, and Arg-CH2R-6, where R ) CH3 (Arg isomer (Figure 9) provided in parentheses).

Figure 12. Comparison of binding orientations, as well as selected B3LYP/6-31G(d) bond lengths (Å) and angles (deg), in (a) neutral and (b) anionic uracil O2(N3) complexes with Arg-CH2R-7 and ArgCH2R-8, where R ) CH3 (Arg isomer (Figure 9) provided in parentheses).

acceptor and the uracil donor. Arg-CH2R-8, on the other hand, involves a nonplanar binding orientation and results in weaker binding to neutral uracil, but stronger binding to the uracil anion due to movement away from the uracil donor site and toward the uracil acceptor. As previously mentioned, the effect of hydrogen bonding on the acidity (∆(acidity)) is directly correlated with the difference between the (large) anionic and (smaller) neutral binding strengths. Therefore, as binding to neutral uracil strengthens, or to the uracil anion weakens, ∆(acidity) decreases. Thus, the

large range in the geometries and binding strengths for the different Arg binding orientations translates into a large range in the effects of hydrogen bonding with Arg on the acidity of uracil. Indeed, depending on the binding orientation, Arg can increase the acidity of uracil by up to 44 kJ mol-1 or decrease the acidity of uracil by up to 27 kJ mol-1. In general, binding orientations involving the terminal amino group as the donor lead to the strongest binding to the uracil anion and therefore the largest increase in the uracil acidity. Alternatively, binding orientations with the imino group as the acceptor lead to the strongest binding to neutral uracil and therefore the smallest increases, as well as the largest decreases, in the acidity of uracil. In summary, Arg has the largest range in binding orientations and effects on the uracil acidity among the amino acids discussed thus far. We note that Arg is likely protonated within enzymatic environments, which will lead to fewer binding orientations (due to the equivalent terminal amino groups), but further enhance the effects of this nucleobase on the acidity of uracil (due to the formal charge). (Vi) Aromatic Groups. (a) Stacking Interactions. Aromatic rings are predominantly considered to interact via π-π stacking. Indeed, computational studies have shown that π-π stacking interactions between the (natural and modified) nucleobases are considerable,31-35,62,65 and therefore potentially as important as hydrogen bonding for the stabilization of DNA helices. Computational studies have also considered stacked aromatic amino acid (Tyr, Trp, His, Phe) dimers,66 and, more recently, the amino acids have been stacked with select (natural and modified) nucleobases.67,68 In the present work, the interactions between the aromatic amino acids and neutral or anionic uracil are investigated to ascertain the influence of π-π interactions on the acidity of uracil. This is important because stacked arrangements of active site amino acids and (nucleobase) substrates have been identified in many enzymes. For example, Phe may interact with uracil using stacking interactions within the active site of uracil DNA glycosylase.21 Table 7 summarizes the optimum MP2/6-31G*(0.25) stacking interactions between uracil or the uracil anion and the aromatic amino acids, where the preferred vertical displacement and angle of rotation for all dimers can be found in the Supporting Information (Table S4).69 Our calculations reveal that the stacking interactions with neutral uracil are up to 30.9 kJ mol-1 and therefore suggest that these interactions have the potential to play important roles in substrate binding and/or identification. However, the stacking interactions with the uracil anion (8-14 kJ mol-1) are much weaker. Therefore, the stacking interactions decrease the acidity of uracil by 10-20 kJ mol-1, and we turn our attention back to hydrogen-bonding interactions.

Acidity of Uracil

Figure 13. Binding orientations, as well as selected B3LYP/6-31G(d) bond lengths (Å) and angles (deg), in neutral uracil O2(N3) complexes with the aromatic amino acids.

(b) Hydrogen-Bonding Interactions. Previous computational work has characterized different types of hydrogen-bonding arrangements available to aromatic amino acids.70 Furthermore, aromatic amino acids often appear in non-stacking orientations within the active sites of enzymes. For example, His may hydrogen bond to the O2 carbonyl of uracil in the active site of uracil DNA glycosylase.21 Indeed, the hydrogen-bonding interactions discussed above for Tyr demonstrate that aromatic amino acids can significantly affect the acidity of uracil. Therefore, the effects of hydrogen bonding with the three other aromatic amino acids (Phe, Trp, His) were investigated. Phe (toluene) primarily hydrogen bonds to uracil in one orientation, where two sp2 C-H bonds share the role of the donor by bridging the uracil carbonyl (Figure 13). Stronger neutral binding and considerably stronger anionic binding is obtained with Phe as compared to CH4. This agrees with previous observations that C-H sp2 donors are stronger than sp3 donors.38 Although Phe produces a smaller effect on the base acidity than Tyr, the effects are still significant in magnitude (9-10 kJ mol-1, Table 8) and larger than those previously discussed for ammonia or methane. It should be noted that a similar binding arrangement can also be found for Tyr (Tyr-2, Figure 13), which leads to slightly smaller effects on the acidity of uracil as compared to Phe (Table 8). Trp (3-methyl-1H-indole) complexes to uracil in two orientations. In Trp-1, the indole nitrogen serves as both the acceptor and the donor (Figure 13). Although this binding orientation is similar to that of ammonia, the neutral binding strengths are weaker (due to weaker N3-H‚‚‚N interactions), and the anionic binding strengths are much stronger, for the Trp complexes. Thus, the effects of Trp on the uracil acidity (45-50 kJ mol-1, Table 8) are much larger than NH3 or the other aromatic amino acids considered thus far. Indeed, the effects of Trp are very close to those discussed for HF. Trp-2 has a binding orientation

J. Phys. Chem. B, Vol. 111, No. 7, 2007 1867 similar to that of uracil as discussed for Phe and Tyr-2,71 although the effects on the acidity are smaller for Trp. A hydrogen can be attached to either the δN1 or the N2 nitrogen in (neutral) histidine, and therefore two histidine models (5-methyl-1H-imidazole and 4-methyl-1H-imidazole) were investigated. Each of these His models coordinates to uracil in an orientation similar to that of Trp-1 (Figure 13). Although the His-1 model produces slightly weaker neutral binding and slightly stronger anionic binding as compared to the His-2 model, the effects of His on the acidity for the two models are within 1 kJ mol-1. Furthermore, the effects of His on the acidity of uracil are approximately 7 kJ mol-1 greater than the effects of Trp-1. Indeed, His leads to among the largest effects due to any amino acid side chain discussed thus far. The greater stabilization provided by His as compared to other amino acids could at least in part explain why histidine coordinates to uracil within the active site of uracil DNA glycosylase. (Vii) The Backbone. In addition to side-chain interactions, DNA-protein complexes have been shown to involve hydrogen bonding with the protein backbone.7,9 Furthermore, the active site of uracil DNA glycosylase shows close contacts between the backbone and the uracil substrate.21 Therefore, we also consider the effects of hydrogen-bonding interactions with the backbone (formylglycinamide).72,73 Although formylglycinamide binds to neutral uracil in a similar orientation (Figure 14) and with a strength (Table 9) similar to that of Asn(Gln)-2, the backbone model leads to significantly stronger anionic binding. Indeed, stronger anionic binding is found for the backbone than any amino acid side chain. The increased binding to the anion arises due to the flexibility of the backbone, which allows significant motion away from the uracil donor and toward the uracil acceptor upon anion formation (Figure 14). The interactions with the backbone increase the acidity of uracil by 2939 kJ mol-1, which is in between the effects of HF and H2O, and less than several side chains. Thus, the effects on the acidity due to interactions with the backbone are significant, and the backbone should be considered in the total picture of active site binding interactions in the future. Environmental Effects on the Enhancement in Uracil Acidity Due to Interactions with Amino Acids. The above discussion illustrates that the acidity of uracil can be increased through interactions with amino acids by up to approximately 60 kJ mol-1 in the gas phase. However, previous studies have shown, for example, that the relative acidity of the N1 and N3 sites in uracil74 and 5-substituted uracil derivatives75 is different in solution as compared to the gas phase. Additionally, previous studies have shown that binding discrete molecules to uracil affects the acid-base properties, and therefore the hydrogenbonding ability, of other sites.22,76 Thus, the environment in biological systems may also change the effects of hydrogenbonding interactions with amino acids on the acidity of uracil. Because the magnitude of this change is not immediately clear, we extend our study by simultaneously accounting for both distinct hydrogen-bonding interactions and the bulk environment, which will allow us to determine the synergy of these effects on the nucleobase acidity. Prior to considering the effects of the environment on the acidity of uracil-amino-acid complexes, the uracil-XH complexes will be considered (Tables 1 and 10). The acidity of uracil increases substantially (by 137.4 kJ mol-1 in ether and 158.1 kJ mol-1 in water) upon solvation (∆(solv), Table 10). Although the acidity of uracil-XH complexes also increases, the magnitude of the enhancement (∆(solv)) is less than that for isolated uracil. The solvent does not stabilize the uracil-XH anionic

1868 J. Phys. Chem. B, Vol. 111, No. 7, 2007

Hunter et al.

TABLE 8: Binding Strengths of Neutral and Anionic Complexes, the N1 Acidity of Complexes, the Effects of Solvation on the Acidity (∆(solv)), and the Effects of Hydrogen Bonding on the Nucleobase Acidity (∆(acidity)) When an Aromatic Amino Acid Is Bound to Uracil in a Variety of Environments (kJ mol-1)a gasb acceptor Phe

Trp-2

His-1

His-2

O2(N3) O4(N3) O4(C5)

2.0 2.3 1.5

11.2 10.9 11.9

1380.3 1380.9 1379.1

9.2 8.6 10.4

1253.0 1251.8 1255.4

127.3 129.1 123.7

-0.9 0.3 -3.3

1211.3 1210.3 1219.5

168.9 170.6 159.7

3.8 4.8 -4.4

O2(N3) O4(N3) O4(C5)

2.6 2.7 1.6

9.4 9.1 9.8

1382.7 1383.1 1381.3

6.8 6.4 8.2

1253.7 1252.0 1256.1

126.6 128.9 123.0

-1.6 0.1 -4.0

1211.5 1210.1 1219.1

168.8 170.8 160.0

3.6 5.0 -4.0

O2(N3) O4(N3) O4(C5)

11.2 16.5 19.1

61.3 58.9 63.2

1339.4 1347.1 1345.4

50.1 42.4 44.1

1228.9 1235.0 1239.0

110.5 112.1 106.4

23.2 17.1 13.1

1195.4 1200.0 1208.6

144.0 147.1 136.8

19.7 15.1 6.5

O2(N3) O4(N3) O4(C5)

3.1 3.0 1.2

5.5 5.2 6.4

1387.1 1387.3 1384.3

2.4 2.2 5.2

1255.8 1254.4 1257.0

124.5 126.5 122.1

-3.7 -2.3 -4.9

1212.9 1211.7 1219.2

167.4 169.2 159.9

2.2 3.4 -4.1

O2(N3) O4(N3) O4(C5)

15.3 17.5 20.8

68.8 66.2 69.9

1336.0 1340.8 1340.4

53.5 48.6 49.1

1227.7 1230.4 1236.4

108.3 110.4 104.0

24.4 21.7 15.7

1198.0 1199.5 1208.4

138.0 141.3 132.0

17.1 15.6 6.7

O2(N3) O4(N3) O4(C5)

16.2 18.3 21.1

68.5 65.9 70.3

1337.2 1341.9 1340.3

52.3 47.6 49.2

1227.8 1230.3 1235.4

109.4 111.6 104.9

24.3 21.8 16.7

1196.7 1197.8 1207.3

140.5 144.1 133.0

18.4 17.3 7.8

CH2, CH2

NH

NH

CH2, CH2

δNH

NH

waterc

neutral BS anion BS acidity ∆(acidity)d acidity ∆(solv)e ∆(acidity)d acidity ∆(solv)e ∆(acidity)d

donor CH2, CH2

Tyr-2

Trp-1

etherc

δNH

NH

a See Figure 1 for depiction of uracil binding sites and Figure 13 for amino acid binding orientations. b Gas-phase B3LYP/6-311+G(2d,p) singlepoint calculations were performed on gas-phase B3LYP/6-31G(d) geometries. c PCM-B3LYP/6-311+G(2d,p) single-point calculations were performed on gas-phase B3LYP/6-31G(d) geometries. d The difference between the acidity of the complex and the acidity of isolated uracil in the same phase. e The difference between the acidity of the complex in solution and in the gas phase.

Figure 14. Binding orientation, as well as selected B3LYP/6-31G(d) bond lengths (Å) and angles (deg), in (a) neutral and (b) anionic uracil O2(N3) complexes with the protein backbone.

complexes as much as the isolated uracil anion because the discrete hydrogen-bonding interactions with the small molecule already provide some stabilization to the uracil anion prior to solvation. Indeed, the stabilization provided by the solvent to the anion (∆(solv)) decreases with an increase in the stabilization provided by the small molecule (i.e., the acidity of XH). Additionally, ∆(solv) increases with the dielectric constant of the solvent. Because solvation stabilizes the uracil anion to a greater extent than the anionic uracil-XH complexes, the effects of discrete hydrogen-bonding interactions on the acidity of uracil (∆(acidity)) are smaller in solution (Table 10) than in the gas phase (Table 1), and systematically decrease with an increase in the dielectric constant of the solvent. However, the magnitude of the reduction in ∆(acidity) depends on the molecule bound. For example, the effect of HF at O2(N3) decreases by approximately 25% in ether (35% in water), while the effect of water at O2(N3) decreases by approximately 50% in ether (65% in water). The larger effect of the solvent on ∆(acidity) for water as compared to HF may occur due to weaker (gas-phase) binding strengths, the smaller gas-phase ∆(acidity), or the binding arrangement, where water forms bidentate hydrogen bonds with uracil, while HF forms strong interactions with only one uracil

site (see Figure 3). Regardless of the reduction in the effects of small molecules on the acidity of uracil, the acidity in different environments can still significantly increase due to discrete hydrogen-bonding interactions, where the largest effect is 38 kJ mol-1 in ether or 34 kJ mol-1 in water. As found for the small molecules, the effects of hydrogen bonding with amino acids in the gas phase decrease when complexes are solvated by ether and decrease further when complexes are solvated by water. However, there is generally a minimum 50% reduction in ∆(acidity) for all amino acids. This is similar to the decreases found for water complexes even though some amino acid complexes have acidities similar to those of hydrogen fluoride complexes. For example, similar to XH ) H2O, the effect of Ser (Thr) at O2(N3) exhibits a 50% decrease upon solvation of the complex by ether and a 55% decrease upon solvation by water. However, the effects of Tyr at O2(N3) also decrease by 55% (65%) in ether (water) despite the fact that the corresponding gas-phase ∆(acidity) is similar to that due to HF. This means that the effects of Tyr in ether and water are much less than the effect of HF. Similarly, Asn(Gln)-1, Asp(Glu)-1, His-1, His-2, and Trp-1 all show effects on the acidity close to HF in the gas phase, and much less than HF in ether and water. We note that these complexes form bidentate binding arrangements, and therefore these findings support our hypothesis that solvents weaken the effects of bidendate hydrogen bonds more significantly than the effects (such as HF) of strong interactions with only one uracil site. Even larger decreases in ∆(acidity) upon solvation are seen for systems with weak (gas-phase) binding. For example, solvation of uracil-Cys complexes decreases the gas-phase ∆(acidity) by 85-90%, which is much more significant than the effects of solvation on uracil-Ser (Thr) complexes (Table 2). Additionally, large decreases in the effects of discrete hydrogen-bonding interactions are noted for complexes with

Acidity of Uracil

J. Phys. Chem. B, Vol. 111, No. 7, 2007 1869

TABLE 9: Binding Strengths of Neutral and Anionic Complexes, the N1 Acidity of Complexes, the Effects of Solvation on the Acidity (∆(solv)), and the Effects of Hydrogen Bonding on the Nucleobase Acidity (∆(acidity)) When the Backbone Is Bound to Uracil in a Variety of Environments (kJ mol-1)a gasb CdO

waterc

neutral BS anion BS acidity ∆(acidity)d acidity ∆(solv)e ∆(acidity)d acidity ∆(solv)e ∆(acidity)d

acceptor donor backbone

etherc

NH O2(N3) O4(N3) O4(C5)

37.7 41.3 33.5

74.0 70.7 72.1

1353.2 1360.0 1350.9

36.3 29.4 38.6

1241.8 1245.7 1239.8

111.4 114.3 111.1

10.3 6.4 12.3

1206.2 1208.0 1209.3

147.0 152.0 141.6

8.9 7.1 5.8

a See Figure 1 for depiction of uracil binding sites and Figure 14 for binding orientation. b Gas-phase B3LYP/6-311+G(2d,p) single-point calculations were performed on gas-phase B3LYP/6-31G(d) geometries. c PCM-B3LYP/6-311+G(2d,p) single-point calculations were performed on gas-phase B3LYP/6-31G(d) geometries. d The difference between the acidity of the complex and the acidity of isolated uracil in the same phase. e The difference between the acidity of the complex in solution and in the gas phase.

TABLE 10: Acidity of the Uracil-XH Complexes (kJ mol-1) Calculated in a Variety of Environmentsa ether O2(N3)

O4(N3)

O4(C5)

HF HF HF H2O H2O H2O NH3 NH3 NH3 CH4 CH4 CH4

water

acidity

∆(solv)b

∆(acidity)c

acidity

∆(solv)b

∆(acidity)c

1252.1 1214.0 1221.3 1225.2 1243.1 1246.4 1243.4 1260.8 1261.8 1249.8 1252.5 1251.6 1256.3

137.4 125.6 127.4 119.4 127.9 129.4 123.2 135.0 136.6 130.8 133.3 134.1 129.3

38.1 30.8 26.9 9.0 5.7 8.7 -8.7 -9.7 2.3 -0.4 0.5 -4.2

1215.1 1181.5 1187.6 1194.4 1208.8 1211.0 1209.7 1222.5 1222.8 1212.5 1212.0 1210.8 1219.6

158.1 161.1 150.2 162.1 164.8 156.9 173.2 175.5 168.1 173.8 174.8 166.0 158.1

33.6 27.5 20.7 6.3 4.1 5.4 -7.5 -7.7 2.6 3.1 4.2 -4.5

a

PCM-B3LYP/6-311+G(2d,p) single-point calculations were performed on gas-phase B3LYP/6-31G(d) geometries. Relative energies include ZPVE and BSSE corrections. b The difference between the acidity of the complex in solution and in the gas phase. c The difference between the acidity of the complex and the acidity of isolated uracil in the same phase.

particularly distorted hydrogen-bonding arrangements. For example, Arg increases the acidity of uracil by up to 44 kJ mol-1 in the gas phase, but only up to 15 (7) kJ mol-1 in ether (water), and the effects of the backbone on the acidity of uracil are also significantly reduced upon solvation. The larger effects of solvation on ∆(acidity) for some amino acid complexes as compared to others mean that some trends in the magnitude of ∆(acidity) with respect to the amino acid bound change upon consideration of the environment. However, some general trends prevail. For example, although the benefits of the aromatic ring in Tyr are smaller in solvation, Tyr leads to larger effects than Ser (or XH ) H2O) in ether and water. Additionally, different binding arrangements lead to different effects on the acidity, where, for example, the acidity of Asn(Gln)-1 is much greater than that of Asn(Gln)-2 and the acidity of Asp(Glu)-1 is less than that of Asp(Glu)-2. Finally, hydrogen bonding with the aromatic amino acids, as well as the backbone, significantly increases the acidity of uracil in solution. In summary, the calculated decreases in the effects of discrete hydrogen-bonding interactions with amino acids on the acidity of uracil are larger than those anticipated on the basis of the small molecule study. This suggests that it is important to consider the effects of the environment in future studies on a case-by-case basis. Nevertheless, discrete interactions with amino acids can significantly increase the acidity of uracil in enzymatic-like and aqueous environments (by up to 30 and 25 kJ mol-1, respectively). Conclusions The present study extends upon our previous investigations of the effects of hydrogen-bonding interactions with small molecules on the acidity of uracil by considering discrete

interactions with amino acids. Our results suggest that, although the small molecules provide an accurate estimate of the effects of hydrogen-bonding interactions on the acidity of uracil in some cases, larger models of other amino acids are required. Consideration of a large range of uracil-amino acid complexes with our most complete models reveals that the effect of interactions with the amino acids on the acidity of uracil is highly dependent upon the binding configuration. We find that the gas-phase acidity of uracil can be increased by up to 60 kJ mol-1 through hydrogen-bonding interactions, while stacking interactions with aromatic amino acids decrease the acidity. Solvation of the uracil complexes was found to decrease the effects of the small molecules and amino acids on the acidity of uracil. However, the magnitude of the effect of the environment varies with the molecule bound, the binding arrangement or geometry, and the strength of the binding interactions in the gas phase. These results hint that it may be important to account for environmental effects on a case-by-case basis in future studies; however, a more detailed investigation of solvent effects is required to more closely consider the dependence on the uracil binding site and dielectric constant. Our calculations show that the effects of discrete interactions with amino acids on the acidity of uracil are significant in enzymatic and aqueous environments (up to 35 and 30 kJ mol-1, respectively). In summary, the present work improves our understanding of the stabilization amino acids can provide to uracil anions. This is particularly important for understanding the role(s) of active site interactions in the mechanism of action of enzymes that catalyze the glycosidic bond cleavage in deoxyuridine, such as uracil DNA glycosylase (UDG), a DNA repair enzyme. Our data show that histidine is among the amino acids with the largest effects on the acidity of uracil in enzymatic environments,

1870 J. Phys. Chem. B, Vol. 111, No. 7, 2007 which may help explain why this amino acid coordinates to the O2 carbonyl of uracil within the active site of UDG. Our results also indicate that UDG active site Asn and backbone residues can provide stabilization to the uracil anion depending on the binding arrangement, and therefore these interactions should be considered in future studies to obtain a complete picture of the stabilization provided by enzymes to nucleobase anions. Because of the large range of binding configurations considered, our results also have more general implications for understanding interactions between amino acids and nucleobases in other systems, as well as the effects of these interactions on an important nucleobase property. Acknowledgment. We would like to thank the Research Corporation, the Natural Sciences and Engineering Research Council of Canada (NSERC), the Canada Foundation for Innovation (CFI), and the New Brunswick Innovation Foundation (NBIF) for financial support. We also gratefully acknowledge the Mount Allison Cluster for Advanced Research (TORCH) for generous allocations of computer resources. Supporting Information Available: Selected B3LYP geometrical parameters for uracil-XH complexes (Figures S1S3), initial stacked dimers used in potential energy surface scans (Figure S4), comparison of uracil-XH acidities calculated with different geometries in the gas phase (Table S1) and ether or water (Table S2), comparison of binding strengths calculated with different Arg models (Table S3), summary of preferred geometry identified from scans of the potential energy surfaces for stacked dimers (Table S4), and Cartesian coordinates for neutral uracil O2(N3) complexes (Table S5). This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Hermann, T. Angew. Chem., Int. Ed. 2000, 39, 1891-1905. (2) Zakrzewska, K.; Lavery, R. Modelling DNA-Protein Interactions; Elsevier: Amsterdam, 1999; Vol. 8. (3) Jones, S.; Daley, D. T. A.; Luscombe, N. M.; Berman, H. M.; Thornton, J. M. Nucleic Acids Res. 2001, 29, 943-954. (4) Allers, J.; Shamoo, Y. J. Mol. Biol. 2001, 311, 75-86. (5) Kim, H.; Jeong, E.; Lee, S.-W.; Han, K. FEBS Lett. 2003, 552, 231-239. (6) Lejeune, D.; Delsaux, N.; Charloteaux, B.; Thomas, A.; Brasseur, R. Proteins: Struct., Funct., Bioinf. 2005, 61, 258-271. (7) Nadassy, K.; Wodak, S. J.; Janin, J. Biochemistry 1999, 38, 19992017. (8) Cheng, A. C.; Chen, W. W.; Fuhrmann, C. N.; Frankel, A. D. J. Mol. Biol. 2003, 327, 781-796. (9) Mandel-Gutfreund, Y.; Schueler, O.; Margalit, H. J. Mol. Biol. 1995, 253, 370-382. (10) Seeman, N. C.; Rosenberg, J. M.; Rich, A. Proc. Natl. Acad. Sci. U.S.A. 1976, 73, 804-808. (11) Matthews, B. W. Nature 1988, 335, 294-295. (12) Kim, K.; Friesner, R. A. J. Am. Chem. Soc. 1997, 119, 1295212961. (13) Hobza, P.; Kabelac, M.; Sponer, J.; Mejzlik, P.; Vondrasek, J. J. Comput. Chem. 1997, 18, 1136-1150. (14) Sponer, J.; Hobza, P. J. Phys. Chem. A 2000, 104, 4592-4597. (15) Alkorta, I.; Elguero, J. J. Phys. Chem. B 2003, 107, 5306-5310. (16) Rozas, I.; Alkorta, I.; Elguero, J. J. Phys. Chem. B 2004, 108, 33353341. (17) Cheng, A. C.; Frankel, A. D. J. Am. Chem. Soc. 2004, 126, 434435. (18) Rozas, I.; Alkorta, I.; Elguero, J. Org. Biomol. Chem. 2005, 3, 366371. (19) For reviews on the DNA glycosylases, see, for example: (a) Dodson, M. L.; Michaels, M. L.; Lloyd, R. S. J. Biol. Chem. 1994, 269, 32709-32712. (b) McCullough, A. K.; Dodson, M. L.; Lloyd, R. S. Annu. ReV. Biochem. 1999, 68, 255-285. (c) Stivers, J. T.; Drohat, A. C. Arch. Biochem. Biophys. 2001, 396, 1-9. (d) Stivers, J. T.; Jiang, Y. L. Chem. ReV. 2003, 103, 2729-2759. (e) Berti, P. J.; McCann, J. A. B. Chem. ReV. 2006, 106, 506-555.

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J. Phys. Chem. B, Vol. 111, No. 7, 2007 1871 6-31G(d,p) level. Although our B3LYP/6-311+G(2d,p) binding strengths reported in Table 2 are smaller than those previously reported, binding strengths similar to Cheng’s are obtained when the smaller (6-31G(d,p)) basis set is implemented. (61) The cis isomer is 22.2 kJ mol-1 more stable than the trans isomer. (62) Hill, G.; Forde, G.; Hill, N.; Lester, J., W. A.; Sokalski, W. A.; Leszczynski, J. Chem. Phys. Lett. 2003, 381, 729-732. (63) Norberg, J.; Foloppe, N.; Nilsson, L. J. Chem. Theory Comput. 2005, 1, 986-993. (64) We note that several other higher-energy minima can be found, which differ in the orientation of the terminal methyl group. (65) (a) Seio, K.; Ukawa, H.; Shohda, K.; Sekine, M. J. Biomol. Struct. Dyn. 2005, 22, 735-746. (b) Wheaton, C. A.; Dobrowolski, S. D.; Millen, A. L.; Wetmore, S. D. Chem. Phys. Lett. 2006, 428, 157-166. (c) Huertas, O.; Blas, J. R.; Soteras, I.; Orozco, M.; Luque, F. J. J. Phys. Chem. A 2006, 110, 510. (66) (a) Kumar, N. V.; Govil, G. Conform. Biol. 1983, 313-321. (b) Kumar, N. V.; Govil, G. Biopolymers 1984, 23, 2009-2024. (c) Kumar, N. V.; Govil, G. Prog. Clin. Biol. Res. 1985, 172, 91-104. (d) Mitchell, J. B. O.; Nandi, C. L.; McDonald, I. K.; Thornton, J. M.; Price, S. L. J. Mol. Biol. 1994, 239, 315-331. (e) Chelli, R.; Gervasio, F. L.; Procacci, P.; Schettino, V. J. Am. Chem. Soc. 2002, 124, 6133-6143. (f) Morozov, A. V.; Misura, K. M. S.; Tsemekhman, K.; Baker, D. J. Phys. Chem. B 2004, 108, 8489-8496. (67) (a) Wintjens, R.; Lievin, J.; Rooman, M.; Buisine, E. J. Mol. Biol. 2000, 302, 395-410. (b) Rooman, M.; Lievin, J.; Buisine, E.; Wintjens, R. J. Mol. Biol. 2002, 319, 67-76. (c) Wintjens, R.; Biot, C.; Rooman, M.; Lievin, J. J. Phys. Chem. A 2003, 107, 6249-6258. (d) Biot, C.; Wintjens, R.; Rooman, M. J. Am. Chem. Soc. 2004, 126, 6220-6221. (e) Cauet, E.; Rooman, M.; Wintjens, R.; Lievin, J.; Biot, C. J. Chem. Theory Comput. 2005, 1, 472-483. (68) Rutledge, L. R.; Campbell-Verduyn, L. S.; Hunter, K. C.; Wetmore, S. D. J. Phys. Chem. B 2006, 110, 19652-19663. (69) We note that the preferred angles of rotation for neutral and anionic uracil are different. Therefore, the binding strengths, and the effect of binding on the acidity, were calculated using both the optimized angle of rotation for the anion (Table S4) and the preferred angle of rotation for neutral uracil. The results for the two data sets generally differ by less than 3 kJ mol-1. (70) Scheiner, S.; Kar, T.; Pattanayak, J. J. Am. Chem. Soc. 2002, 124, 13257-13264. (71) We note that similar binding arrangements may also be possible with other sp2 C-H donors within the Trp ring; however, we expect the results to be similar to Trp-2, and therefore we do not further pursue these binding arrangements. (72) We note that Alkorta et al. (ref 15) modeled the protein backbone as N-formylglycinamide in the β and γ conformations. However, because the β conformation must be optimized in fixed Cs symmetry, where release of this constraint leads to the γ conformation, we only consider the γ conformation. (73) Although a binding arrangement similar to that discussed for Asn(Gln)-2 (Figure 5) can be found for the backbone model, we do not consider this interaction in the present work because this binding cannot occur with the true conformation of the protein backbone. (74) Kurinovich, M. A.; Lee, J. K. J. Am. Chem. Soc. 2000, 122, 62586262. (75) Jang, Y. H.; Sowers, L. C.; Cagin, T.; Goddard, W. A., III. J. Phys. Chem. A 2001, 105, 274-280. (76) Nguyen, M. T.; Chandra, A. K.; Zeegers-Huyskens, T. J. Chem. Soc., Faraday Trans. 1998, 94, 1277-1280.