Effects of Hydration on the Zwitterion Trialanine Conformation by

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The Effects of Hydration on the Zwitterion Trialanine Conformation by Electronic Structure Theory Giuseppe Lanza, and Maria A. Chiacchio J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b08108 • Publication Date (Web): 21 Oct 2016 Downloaded from http://pubs.acs.org on October 24, 2016

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The Journal of Physical Chemistry

The Effects of Hydration on the Zwitterion Trialanine Conformation by Electronic Structure Theory Giuseppe Lanza* and Maria A. Chiacchio Dipartimento di Scienze del Farmaco, Università di Catania, Viale A. Doria 6, 95125 Catania (Italy)

ABSTRACT

Exploration of interfacial hydration networks of zwitterion and non-ionized trialanine has been performed using DFT-M062X quantum chemical computations explicitly considering up to 41 water molecules. The step-by-step water molecules peptide surrounding, carried out for unfolded extended (β), polyproline II (PPII) conformations reveals the crucial importance of explicit solvent effects in stabilizing the zwitterion form and the left-handed PPII-helix ubiquitously found at room temperature for short polyalanines. Hydration effects are much greater for the ionized form of the peptide, thus the zwitterion is about 10 kcal mol-1 more stable than the nonionized form. For the β→PPII transformation, the two components of free Gibbs energy act in the opposite direction, thus it is favored by enthalpy but not by entropy. These findings agree with experimental data which report an equilibrium between these conformers modulated by

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temperature. Thermodynamic functions of the four conformers (β-β, β-PPII, PPII-β and PPIIPPII) for zwitterion trialanine are similar to those derived for the protonated one (Ala3H+); therefore peptidic conformation is independent of the pH of the solution. Rather, it reflects the high propensity of alanine towards PPII helix. The enthalpic preference of the PPII has electrostatic origin and it is owing to a more favorable interaction of dipole of each peptidic residue with water dipole of H-bonded molecules.

1. INTRODUCTION The elucidation of the water-biomolecule interactions is one of the most important areas of modern science and many major journals have recently dedicated issues to this topic.1-6 In fact, environmental effects play a key role in all biological processes and hence they are an essential ingredient for the development of wide spread methods for practical applications such as research in pharmaceutical science.7 To this purpose, a tremendous number of predictive hydration models has been developed and, although many aspects have been clarified over the years other issues remain unraveled and new questions have arisen. To review computation methods that evaluate solvent effects is something hopeless, but adopted strategies are decidedly limited in number. The Quantitative Structure Property Relationship and Linear Solvation Energy Relationship "top-down" strategies found large applications in pharmaceutical science for drug development.89

These provide reasonable results for a group of similar molecules; however, since they adopt a

modest representation of the interactions at the molecular level, results are not transferable to other systems.


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In the implicit solvent models, derived from Born and Onsager models, the solute is immerged in a solvent represented as a continuous medium.10 In the accessible surface area methods the interaction energy is taken proportional to the accessible surface area of each atom forming the molecule.11 These are useful to describe hydrophobic effects but it gives a poor description of electrostatic contributions. The implicit methods the interactions of solute charge distribution and homogeneous polarizable solvent are taken into account. This class of methods includes the quantum chemistry well known polarizable continuum model (PCM),12 the SMx continuum models,13 and the conductor-like screening model (COSMO).14 In molecular mechanics computations implicit solvent effects are generally also modeled by methods derived from the Poisson-Boltzmann equation.15 The reference interaction site model (RISM)16 and Langevin dipole model,17 in addition to the contributions of implicit solvation consider some specific effects. In the RISM method, the solute can be treated both quantum mechanically or by force fields, while the solvent molecules are represented by classical statistical mechanics based on the assumption of infinite dilution of the solute molecule. To this aim, the integral equation theory of liquids provides the ensemble of solvent configurations. In the Langevin dipole model solvent molecules are represented by point dipoles placed on a cubic grid. According to the Langevin expression, the electrostatic field of the solute reorients and polarizes the grid point dipoles. Explicit water modeling in molecular mechanics and Monte Carlo simulations with empirical force fields are routinely used.18-20 In principle all specific and non-specific solute-solvent contributions are considered and the energy of a molecule is computed in terms of a simple function which accounts for distortion from “ideal” bond distances and angle. Of course, explicit methods account for the short range interactions however, depending on the number of water


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molecules considered, it might be useful to combine it with the implicit model to account for long-range electrostatic interactions. A significant improvement of the solute-solvent description is gathered with the mixed quantum mechanics and molecular mechanics schemes (QM/MM).21 In these methods, the solute and some water molecules or the region of the solute of interest are treated at an appropriate level of quantum chemistry theory, while the remainder is described by a molecular mechanics force field. The Car-Parrinello molecular dynamics simulations treat nuclear motions in a classical Newtonian way while electronic motions are quantum mechanically treated.22 Electronic wave functions and charge densities, expanded in a plane-wave basis set, allow for reliable and promising applications to biomolecular chemistry. The supermolecule model, known also as the cluster approach, is the "most chemical" of all methods to account for the hydration occurrence of a given solute. Generally, the hydration-shell around a molecule is constructed gradually, increasing the number of molecules placed on the contact surface, in a bottom-up fashion. Several biomolecules have been studied with this approach since quantum chemical codes have been developed during the early 1970s.23-24 In spite of the substantial results obtained at that time, the supermolecule approach did not receive a great deal of attention in subsequent years during the computers/quantum chemical codes boom.12 At first sight, this method does not seem to give an exhaustive physical description of the whole solute-solvent system. However, some aspects of fundamental importance make it appealing and complementary/alternative to all the other methods. On the top of these features there is undoubtedly the correct quantum mechanics description of the water-peptide and water-water


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molecules interactions. In principle there is no limit to the accuracy obtainable in quantum chemical calculations and reliable DFT, MP2 or CCSD(T) allow the very best electronic structure description. Practically, today computers allow us to study, in a reasonable time, systems up to 200 and 100 atoms at DFT and MP2, respectively, with a medium size basis sets. Although we are far from the number of atoms constituting the biologically significant systems, the absence of empirical parameters allow for the assessment of physical-chemical characteristics a without prejudice basis. Recently, we clarified a way to construct "chemically logical" supermolecules which, combined with high level quantum chemical methodologies, allowed for a fair representation of the molecular environment around the AcAlaNH2 and Ala3H+ peptides.25-27 Water molecules were able to make explicit hydrogen bonds with polar groups of peptides, aligning dipole optimally and staying away from the hydrophobic groups. Hydrophilic interactions are particularly strong and their action is not limited to rendering peptide soluble and stabilizing ionized forms in water rather, they also modulate the peptide chain conformation. Even though our model partially account for hydrophobic interactions, the fruitful experimental/theoretical data comparison on the relative stability of polyproline II (PPII) and extended (β) suggest that hydrophilic interactions have a prominent role in driving peptidic chain conformation for analyzed peptides. This is in agreement with the recent view about relative importance of hydrophilic vs. hydrophobic interactions proposed by Ben-Naim: "contrary to the almost universal belief that hydrophobic interactions are the most important driving forces in biochemical forces, I believe that hydrophilic interactions are far more important in biochemistry".28


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In the present contribution, we report a quantum chemical study on hydration phenomena at zwitterion and non-ionized forms of the neutral alanine tripeptide bonded to several water molecules Ala3⋅nH2O (n=2-41). The explicit model coupled with the implicit approach allows us to quantify, quantum-chemically, the differential hydration energy in the Ala3 peptide with terminal charged groups and neutral termini on the fully-extended chain conformation (β-β). This amount is responsible for the greater stability of the zwitterionic form of amino acids and peptides in water contrary to what happens in the gas phase.29 Furthermore, for the zwitterion species other three conformations have been identified experimentally in equilibrium, the "pure" polyproline (PPII-PPII) and the "mixed" β-PPII / PPII-β ones whilr the α-helix seems to have a negligeable population.30-32 This study will allow us to verify the validity of our "chemical logical" approach to another peptide, which despite its being similar to the already studied Ala3H+ has a significant difference at the ends of the chain. For example, in the recent years there was a debate on the effect of pH on the peptide conformation, i.e. if structural changes occur when unblocked tripeptides adopt cation, zwitterion or anion forms.31,33 Furthermore, a simple and rationale model to explain the high propensity of short polyalanines to adopt PPII conformation is proposed on the basis of electronic structure. 2. CALCULATION METHODS All computations were performed at the density functional theory with the M06-2X, i.e. a hybrid meta-generalized gradient approximation functional.34 The M06-2X has the 54% HartreeFock exchange and its reliability is well established and its good computational performance allowed us to treat systems with as many as 156 atoms with 4899 basis functions (in the Ala3⋅41H2O complex).


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Geometries were optimized with the 6-31+G* basis set and included implicitly solvent effects. Minima were characterized evaluating the hessian matrix and the associated harmonic vibrational frequencies. The polarization effects of the peptide-water were considered using the polarized continuum method (PCM) adopting a 78.36 dielectric constant for water as implemented in the G09 program.35 To improve energetics and to reduce intermolecular basis set superposition error, single point energy at the optimized geometry was performed using the more accurate aug-cc-pVTZ basis sets including implicit solvent effects. To calculate the entropy, S°298, the different contributions to the partition function were evaluated by using the standard expressions for an ideal gas in the canonical ensemble, the harmonic oscillator, and the rigid rotor approximations. For selected cases, the computed electronic energies were corrected for zero-point vibrational and thermal contributions to obtain standard enthalpy changes and Gibbs free energy at 298 K (∆H°298 and ∆G°298). The grid mesh in integral evaluation was settled to the "Integral(UltraFineGrid)" option because vibrational frequencies and related zero-point energy and absolute entropy are sensitive to integral accuracy.27 Partial atomic charges have been evaluated by means of the Charge Model 5 (CM5), an extension of Hirshfeld population analysis.36,37 3. HYDRATION MODEL FORMULATION The construction of a hydration shell was carried out by adding water molecules step-by-step, until a complete hydrogen-bonded water network surrounds the peptide. This approach can allow us: i) to gather useful information on specific peptide-water and water-water interactions, ii) to


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estimate the convergence of peptide molecular properties in function of the size of the water cluster, and iii) to derive a minimal model for the very first hydration sphere around the peptide.25-27 The rigorous application of such a procedure would require a global energy minimization of the considered models, as already reported for hydration of the alanine amino acid.38 However, hydration with a small number of water molecules results in the formation of a “water droplet” with the amino acid bonded to its surface i.e., a partial hydrate alanine. A fully-hydrated amino acid as the global minimum was obtained when a large number of water molecules (greater than 46) was considered.38 Of course, for the present trialanine, hundreds of water molecules would be necessary to get the global minimum with a full hydrated peptide. Therefore, if we try to follow this route, the use of very reliable quantum chemical methods becomes prohibitive and the opportunity to employ the supermolecule approach on large scale (in terms of peptide size) is hampered. To overcome these difficulties, the handcraft construction of the H-bond network was followed. With the aim to use a minimal number of water molecules to hydrate the peptide, the geometries of Ala3⋅nH2O models were generated using well known structural features of waterwater and peptide-water H-bonding and a primary energetic criterion: the maximization of the peptide-water interaction energy, ∆Eint (eq. 1), i.e., the difference between the total electronic energies of isolated water cluster/bare peptide and their assembly complex at the peptide-water optimized geometry (eq. 1). Ala3 + (H2O)n → Ala3·nH2O

∆Eint

(1)


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The model formation energy, ∆Eform, is computed as the difference of electronic energy of reagents (bare Ala3 peptide with extended conformation) and the peptide-water complex at their respective minima of energy. Ala3 + nH2O → Ala3·nH2O

∆Eform

(2)

The ∆Eform accounts for both peptide-water and water-water forces, and it provides an estimation of conformer relative stability for each set of structures with the same number of water molecules. For peptide–water complexes with few solvent molecules, the ∆Eform and ∆Eint follow the same trend, the more stable structure corresponds to the more hydrated. As the number of solvent molecules increase (n>4), the global minimum is obtained arranging water units over the already formed water cluster in a region in which there is no contact with the peptide.25 These structures are useless for our purpose, thus the maximization of the ∆Eint criterion becomes an essential requisite for quantum chemical applications to hydration of biochemical significant oligopeptides. The criteria to draw initial structures for energy minimization are derived from well-known structural features. Each water molecule forms two H-bonds as an acceptor and two as a donor, amide groups form two H-bonds as an acceptor (the oxygen of carbonyl group) and one as donor (>NH group). There is a strong synergy among donor/acceptor H-bonds and the best energy match is reached when a network of alternation acceptor and donor H-bonds takes place. Water molecules surround the peptide maximizing the number of H-bonds and their strength to realize the highest density packing. Furthermore, an H-bond is highly directional with the D-H···A bond angle close to the linearity.25-27


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Figure 1. Schematic H-bond network of water molecules over zwitterion Ala3 with the dipole of solvent particles aligned in opposite direction to peptide macrodipole.

These rules have been described in more details in our previous reports. However, another characteristic that is worthy of note for the water network construction around zwitterion peptide is the dipole alignment. Because of the charged termini, the Ala3 zwitterion shows a huge dipole moment (Figure 1) that introduces an additional constraint for solvent molecules orientation in wires and clusters of water molecules in contact with the peptide. The most favorable energy is reached when peptide and water dipoles have opposite directions as schematically reported in Figure 1, the explicit water molecule polarization. This coupling reduces the dipole of peptidewater models as the number of water bonded increase and progressively increase hydration energy. Considering all mentioned criteria a large number of Ala3·nH2O (n=2 to 41) models with peptide in various conformations and water molecules in various configurations (Table 1) were optimized to estimate molecular properties and to elucidate the main factors that govern the conformation preference. Among considered models a particular attention has been devoted to


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the zwitterion Ala3·22H2O adduct, since twenty-two water molecules is the lowest number necessary to hydrate the four unfolded conformations of peptide without significant structural constraints. Note that there are very different practical difficulties in building hydration sphere around various peptide conformations. The almost two-dimensional backbone chain of the β-β conformer allows a very easy construction of water networks around the peptide and few attempts were necessary. We were able to increase the number of water molecules up to 41 with reasonable efforts. For the other cases and in particular for the PPII-PPII conformation, several attempts were necessary to get suitable H-bond networks. The three-dimensional extension of the peptidic chain makes evaluation of the water network much more laborious and, because of the more compact form, the water molecules have a great propensity to cluster together during the geometry optimization with the consequent lack of some peptide-water H-bond. Table 1. Number of optimized structures of Ala3⋅nH2O models for each conformation of peptide. Number of water β-β molecules

β-β_non-ionized

β-PPII

PPII-β

PPII-PPII

α

2

1

1

1

1

1

2

4

1

1

1

1

1

3

9

3

3

1

1

1

3

15

1

1

2

2

2

3

18

1

1

2

1

2

19

1

20

2

22

3

1

3

3

3

24

1

1

27, 32, 38 and 41

1


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4. RESULTS AND DISCUSSION 4.1. Bare peptide Geometry Optimizations. The neutral Ala3 peptide can exist in two different chemical forms, the zwitterion and the non-ionized; therefore, both Born-Oppenhimer surfaces should be scrutinized. Nevertheless, we know that in aqueous solution at the isoelectric point (pH∼5.5) the zwitterion is exclusively present, hence its conformations will be the main object of the present work. At the same time, to shed light on differential hydration of the two forms, calculations on the fully extended conformation of the non-ionized species were also carried out. The bare zwitterion reveals the presence of four minima for the unfolded structure corresponding to “pure” and “mixed” extended and polyproline II in about 3 kcal mol-1 energy range (Figure S1). At lower energy (-2.6 / -4.7 kcal mol-1) there are three compact zwitterion structures which show a strong H-bond between the two terminally charged -NH3+ and -CO2groups with a reversed C11 enclosure. Dihedral angles of two of them do not quote any structural motif and they are indicated as C11_A and C11_B while the third has φCentral=-58.3° and ψ Central=-35.2° that are close to those expected for α-helix and hereafter it is indicated as α. The non-ionized species have also been found

lower in energy than the β -β β zwitterion

conformer (-5.4 and -7.2 kcal mol-1 for β -β β_non-ionized and α_non-ionized, respectively). It is clear that unfolded β and PPII conformations of trialanine zwitterion, ubiquitously found in experiments, are due to the explicit water coordination to the peptide. 4.2. Hydrating the zwitterion β-β conformer. The building-up "hydration-shell" can be started both from the -NH3+ or -CO2- terminals since dispersion of charges have prominent roles in the hydration of the peptide. The hydration of the -NH3+ group is much easier and intuitive to


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be developed, since each hydrogen atom of the −NH3+ group coordinates the oxygen of a water molecule. Therefore, one or two couples of water molecules can be added to connect the -NH3+ group and the adjacent >CO in the “up” region, satisfying the alternation and the linearity of Hbonding criteria (2H2O_β-β and 4H2O_β-β structures in Figures 2 and S2). These structures show the formation of two H-bonds between peptide and water molecules and one water-water H-bond for each (H2O)2 bridge, which results in a noticeable electronic energy of formation, 16.1 and -30.6 kcal mol-1 for 2H2O_β-β and 4H2O_β-β, respectively.

Figure 2. Optimized molecular structures of Ala3⋅nH2O (n=4-41) complexes with the peptide in the zwitterion form and β-β conformation. Formation energies (kcal mol-1) at the 6-31+G* (value on the left) and aug-cc-pVTZ (value on the right) were computed relative to the Ala3 (in the β-β conformation) and “n” isolated water molecules. Other structures are reported in Figure S2.


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The successive water molecule coordination involves the two (H2O)2 bridges bonded to the Nterminus and the >N-H and -CO2- groups of the C-terminal residue. Various attempts were performed and the best choice seems to be the formation of a network of nine water molecules in the "up" region and three minima for the 9H2O_β-β model were found (Figures 2 and S2). In the 9H2O_β-β_A and 9H2O_β-β_B models two water molecules coordinate the -CO2- terminal and differ for the water molecule arrangement over the N-terminal residue. In particular, the 9H2O_β-β_B model has an extra water-water H-bond that infers a greater energetic stability (1.2 kcal mol-1). However, this extra water-water H-bond implies a greater rigidity of the water assembly, the peptide-water contacts on the C-terminal residue of the 9H2O_β-β_B model are longer than 9H2O_β-β_A model. This indicates a less favorable structural peptide-water match and hence a less exoenergetic hydration energy for the 9H2O_β-β_B model (∼ 5 kcal mol-1). The 9H2O_β-β_C model shows a single water molecule coordinated to the -CO2- group. The >NH--OH2 and the -CO2----H2O bond distances over the C-terminal residue suggest a good structural match. Nevertheless, the lack of a -CO2---H2O H-bond renders this model unfavorable, both in terms of ∆Eform and ∆Eint energies with respect to 9H2O_β-β_A. The long >NH---OH2 distance and the large deviation from linearity of the >N-H--OH2 and of the two -COO---H2O bond angles suggest that water coordination over C-terminal would require additional modeling improvements. However, this will be addressed later once all other polar groups are hydrated. Six water molecules in the "down" region cover polar groups of N-terminal and central residues. Judging from the peptide-water H-bond distances and bond angles, the 15H2O_β-β does not present structural strain and we can go ahead in completing the -CO2- hydration. The 18H2O_β-β allows for the coordination of one water molecule to the oxygen of the carboxyl in


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the "down" region, while the 19H2O_β-β and 20H2O_β-β models complete polar groups hydration and the -CO2- moiety with four H-bonded water molecules. The H-bond lengths of added water molecules and related H-bond angles indicate the 20H2O_β-β as a better model and in fact, while having the same water-peptide H-bonds of the 19H2O_β-β one, the former is more exoenergetic in terms of hydration energy (-85.9 vs. -87.1). The building-up "hydration-shell" continues by adding two molecules in the "up" region close to the oxygen of the carboxylic group, 22H2O. The three considered models differ for the water cluster arrangement over the oxygen of the carboxylic group in the "up" region. Looking at the peptide-water contacts it is evident that the 22H2O_β-β_B and 22H2O_β-β_C overcome the constraints discussed about the 9H2O_β-β models and, thus, they are energetically favored. These structures seem to be the best geometries for the minimal hydration; however, it is clear that much more water molecules are necessary for charge dispersion of this zwitterion. Further water molecules were added to connect dangling H-bonds of solvent molecules placed on the “up” and on the “down” regions of the minimal hydrated 22H2O_β-β_B, thus the 24H2O_β-β and 27H2O_β-β were constructed (Figure S2). To improve charge dispersion, other five molecules were bonded to the water cluster over the -CO2- group (32H2O_β-β), other six water molecules were bonded to the group surrounding the terminal charged amine (38H2O_β-β), then another three water molecules have been placed on the -CO2- side (41H2O_β-β). The addition of 19 water molecules to the 22H2O_β-β_B minimal model improves the description of hydration phenomena but does not significantly alter the structure of the peptide as a result of the dihedral angle values (Table S1). 4.3. Hydrating the non-ionized β-β conformer. The building-up "hydration-shell" above the non-ionized peptide was pursued similarly to the zwitterion form and the 2H2O_β-β_nonionized, 4H2O_β-β_non-ionized, 9H2O_β-β_non-ionized_A, 9H2O_β-β_non-ionized_B and


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9H2O_β-β_non-ionized_C structures have similar shapes (Figure 3). However, the waterpeptide H-bonds which involve peptide termini in the non-ionized form are much weaker with bond lengths about 0.2 Å longer than the corresponding ones in the zwitterion and in the case of the 9H2O_β-β_non-ionized_B model one >NH---OH2 disappear. In this case the (H2O)5 cluster above the N-terminal residue reaches a better structure, water-water interactions are maximized and the 9H2O_β-β_non-ionized_B model turns out to be the most stable in spite of the lack of one peptide-water H-bond. The weakening or disappearance of water - peptide H-bonds at the ends of the non-ionized peptide reflect the huge contribution of ion-dipole interactions in the hydration of the charged groups of the zwitterion. The successive model 15H2O_β-β_non-ionized was derived from the 9H2O_β-β_nonionized_C and differs from corresponding zwitterion for two features. Firstly, a water molecule from the "up" region is moved in the "down" because it is unnecessary to construct minimal hydration. Secondly, the lone pair of the terminal amine located in the "down" region acts as a strong H-bond acceptor with the -H2N---HOH bond length of 1.70 Å. Water-peptide modeling goes on completing carboxyl group hydratation on the "up" region (18H2O_β-β_non-ionized) with the formation of a five-member water cluster above the carboxyl group and then on the "down" region (22H2O_β-β_non-ionized). To form the 24H2O_β-β_non-ionized model, two water molecules were added to link dangling H-bonds of solvent units placed on the “up” and on the “down” regions of the minimal hydrated 22H2O_β-β_non-ionized.


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Figure 3. Optimized molecular structures of Ala3⋅nH2O (n=2-24) complexes with the peptide in the non-ionized form and β-β conformation. Formation energies (kcal mol-1) at the 6-31+G* (value on the left) and aug-cc-pVTZ (value on the right) were computed relative to the Ala3 zwitterion (in the β-β conformation) and “n” isolated water molecules.

The hydration electronic energy, ∆Eint, for both zwitterion and non-ionized peptide-water complexes with the peptidic chain in the fully extended arrangement are reported in Figure 4. For both zwitterion and non-ionized forms, the ∆Einter becomes more negative (more exoenergetic) as the number of hydrating molecules increases because of the increase in number of peptide-water


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formed H-bonds. Both data for zwitterion and non-ionized distribute approximately on rectangular hyperbola with asymptotic values of about -103 and -80 kcal mol-1, respectively. The curve for non-ionized species does not show significant energy change for a model that has more than twenty-two waters because no further H-bonds are formed thus, in this case the waterpeptide H-bonding is the driving interaction. Instead, the curve for zwitterion becomes more exoenergetic even for a model greater than twenty-two water molecules up to 41 even though no further water-peptide H-bonds are formed. Long range solute-solvent forces are still active and that go beyond the very first contact water molecules. In this context, we observe that the huge dipole of the bare zwitterion β-β conformer (42.1 D) significantly reduces upon the number of water molecules increase and for the 41H2O_β β-β β model it is 14.8 D. The explicit water molecule polarization illustrations in Figure 1 give a crucial contribution to the overall hydration phenomena. A similar situation has already been reported for the protonated trialanine, Ala3H+, which shows an intermediate ionized state with an estimated hydration energy of about -90 kcal mol-1. The differential effect of explicit water coordination reverses the initial, bare peptide, electronic energy stability of the non-ionized and zwitterion forms, being the latter computed 7.3 and 9.3 kcal mol-1 lower in energy for the 22H2O and 24H2O models, respectively (Figures 2, S2 and 3). The zero-point energy and thermal corrections slightly reduce zwitterion stability (∆H°298= 6.3 and 8.1 kcal mol-1). Even though these values are not close to the convergence with respect to the number of coordinating water molecules, they are similar to computed and experimental values reported for alanine (10-12 kcal mol-1) and glycine (10.3 kcal mol-1), respectively.38,39


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Figure 4. Calculated M06-2X/aug-cc-pVTZ/PCM=water hydration energies (∆Eint, solid lines) for the Ala3⋅nH2O complexes with the peptide in the β-β conformation in both zwitterion and non-ionized forms. The dashed line shows the relative energy at the respective optimized geometry (Enon-ionized - Ezwitterion).

All these findings are in agreement with the general view that the environment has important differential effects on zwitterion and non-ionized peptide forms and actually hydration is the major contribution to stabilizing charged groups.


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Figure 5. Optimized molecular structures of Ala3⋅nH2O (n=2-22) complexes with the peptide in the zwitterion form and β-PPII conformation. Formation energies (kcal mol-1) at the 6-31+G* (value on the left) and aug-cc-pVTZ (value on the right) were computed relative to the Ala3 zwitterion (in the β-β conformation) and “n” isolated water molecules.

4.4. Hydrating the zwitterion β-PPII conformer. Because of the geometrical analogies with the zwitterion β-β conformer, the hydration-shell construction occurs in a similar way with the formation of 2H2O_β-PPII, 4H2O_β-PPII and 9H2O_β-PPII (Figure 5). The nine water molecule arrangement resembles the 9H2O_β-β_C with the -CO2- involved only in one H-bond. The hydration in the "down" region does not lead to the coordination of two water molecules to the >CO of the central amino acid. Instead there is also the involvement of the charged carboxyl.


20

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Two structures were found depending if the two water molecules bonded to the carboxyl group coordinate the same oxygen (15H2O_β-PPII_A) or coordinate both the oxygens (15H2O_βPPII_B). The 18H2O_β-PPII models complete hydration of the central >CO and the carboxyl has three water molecules coordinated. The structures and energies of the 18H2O_β-PPII_A and 18H2O_β-PPII_A' are very similar and show a substantial gain in energy with respect to the analogue 18H2O_β-β model (about -7 kcal mol-1), thus indicating the progressive more efficient hydration of the PPII part of the peptide. Four additional water molecules placed in the region between the >NH and -CO2- of the Cterminal residue, connect the “top” and “down” water layers realizing the minimal hydration of the peptide. The 22H2O_β-PPII_A, 22H2O_β-PPII_A', and 22H2O_β-PPII_B have a similar energy (within 1 kcal mol-1) because they have a similar water covering.

4.5. Hydrating the zwitterion PPII-β conformer. Conversely to what was found for the 4H2O_β-β and 4H2O_β-PPII models, the 4H2O_PPII-β geometry optimization leads invariably to the formation of a stronger H-bond with the >CO group of the central residue (Figure 6). However, the two H-bonds on the >CO of the N−terminal residue are restored in the 9H2O_PPII-β model. Adding other six water molecules, we got the 15H2O_PPII-β_A and the 15H2O_PPII-β_B models depending if the two coordinating water molecules to the carboxyl group bond the same oxygen (15H2O_PPII-β_A) or both oxygens (15H2O_PPII-β_B). Various structures can be formed for the 18H2O_PPII-β model; however what is reported in Figure 6 is the most stable since it involves the coordination of four water molecules over the terminal


21


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carboxyl group. Although the microsolvation of all hydrophilic groups is completed, the addition of four water molecules close to the C-terminal, the 22H2O_PPII-β models, removes some structural strain and allow for more exhaustive coulombic interactions. The three models have peptide-water interaction energy very similar (∆Eint ∼ -95 kcal mol-1), because they have the same number of peptide-water H-bonds. However, the 22H2O_PPII-β_C and 22H2O_PPII-β_B models have a more compact water-water network with two and one additional H-bonds, respectively. This infers a greater overall stability.

Figure 6. Optimized molecular structures and formation energies (kcal mol-1) of the Ala3⋅nH2O (n=2-22) complexes with the peptide in the zwitterion form and PPII-β conformation.


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Figure 7. Optimized molecular structures and formation energies (kcal mol-1) of the Ala3⋅nH2O (n=2-22) complexes with the peptide in the zwitterion form and PPII-PPII conformation.

4.6. Hydrating the zwitterion PPII-PPII conformer. The initial microsolvation around the PPII-PPII conformer occurs in a similar way to that found for the PPII-β one with the involvement of the >CO of the central residue for the 4H2O_PPII-PPII model (Figure 7). The 9H2O_PPII-PPII model involves both oxygens of carboxyl group in H-bonding, conversely to what was observed for the 9H2O_PPII-β model where two water molecules coordinate the same oxygen atom. The 15H2O_PPII-PPII_A and 15H2O_PPII-PPII_B models mainly differ for the number of coordinating water molecules to the carboxylic group, three and two respectively. The lack of the peptide-water H-bond is compensated by the formation of an extra water-water H-


23


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bond, thus the two structures are close in energy. With the addition of three water molecules over the carboxylic group, the 18H2O_PPII-PPII models, all hydrophilic groups of peptide are hydrated, thus for compact structures like PPII, a lower number of water molecules is necessary to reach the minimal hydration. Nevertheless, four water molecules of water were added in the region close to the carboxylic group to improve charge dispersion and H-bonding connectivity (22H2O_PPII-PPII). The 22H2O_PPII-PPII_A' structure was constructed with the carboxyl group accepting five H-bonds. Despite the fact that it has one more H-bond than the 22H2O_PPII-PPII_A one, it is slightly less stable, hence the assumption that -CO2- accepts four H-bonds, once again adopted in the present study, is an appropriate choice. 4.7. Hydrating the zwitterion α conformer. The four folded structures (C11_A, C11_B, α, α_non-ionized) show a strong H-bond between termini which infer them a great stability. However, when a water molecule is placed in the proximity of this H-bond, it spontaneously inserts itself between termini and two more efficient H-bonds are formed with the water molecule. These bare structures, although more stable than β and PPII ones, cannot exist in aqueous solutions. Instead, they can play some role when the peptide is in the gas-phase.40-41 Among the four folded structures, the zwitterion α-helix is worthy of further investigation since it is predicted to occur significantly in molecular dynamics simulations.42-43 The coordination of two water molecules between the termini of the peptide leads to the formation of the 2H2O_α α_A structure, without any intrapeptide H-bond, and of the 2H2O_α α_B structure, which maintains the intrapeptide NH3---O2C H-bond (Figure 8). Both structures show four H-bonds and have a comparable stability. The 4H2O_α α_A and the 4H2O_α α_B form two water molecule bridges (3+1 and 2+2 solvent units, respectively) which involve the same oxygen of the terminal CO2- group while for the 4H2O_α α_C the 3+1 water molecule bridges involve both the oxygens of the


24

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carboxyl. Adding one water molecule close to a water chain of the 4H2O_α α_A and 4H2O_α α_B models to bond the second oxygen of the carboxyl, and adding a chain of four water molecules over the free hydrogen of the -NH3+ group and >C=O groups of the N-terminal and central residues, the 9H2O_α α_A and the 9H2O_α α_B are obtained. The 9H2O_α α_C was formed enlarging a bridge (3+2) that directly connects the terminal NH3+ and CO2- and adding a chain of three water molecules over the free hydrogen of the -NH3+ group and >C=O groups of the Nterminal and central residues. A large series of calculations was performed also for the α-helix with fifteen bonded water molecules and found minima are reported in Figure 8. In these structures, all polar groups of the peptide form the optimal number of H-bonds with water molecules and, because of the compact shape of the α-helix, the minimal hydration sphere is already reached and no further model improvement is necessary.

The most important structural hint that comes from all the 9H2O_α α and 15H2O_α α models is the proximity of the N-H bonds of central and C-terminal residues, which are in a region distal to the C=O bonds of N-terminal and central residues. The alternance of H-bond donors (N-H) and acceptors (C=O) of an adjacent peptidic bonds is lost, hence the synergy of H-bonds is reduced and peptide-water interactions are less efficient than those active in unfolded models.24 Indeed, the formation energy of 9H2O_α α and 15H2O_α α is almost comparable to those of the 9H2O_PPII-PPII and 15H2O_PPII-PPII models despite the fact that at this stage of modeling the latter structures are partially hydrated and have several peptide-water H-bonds to be formed.


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Figure 8. Optimized molecular structures and formation energies (kcal mol-1) of the Ala3⋅nH2O (n=2-15) complexes with the peptide in the zwitterion form and α-helix conformation. The α-helix models are also disfavored by the entropy, and in fact, all models presently analyzed have the lowest values of S°298, that for the 15H2O_α α case produce the -T∆S βcontribution to ∆G of about 3 and 9 kcal mol-1 with respect to 15H2O_PPII-PPII and 15H2O_β β models. The partial synergy lacking in H-bonding formation and the loss of conformational entropy upon folding suggest that α-helix population is predicted to be insignificant for the trialanine.25 In this context, it is useful to remember that α-helix stabilization is not owing to polar groups microhydration, rather it is ascribed to intrapeptide H-bonding in the C13 enclosure and to the related huge macrodipole which is involved in a strong nonspecific interaction with


26

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the solvent.44 The Ala3 is too short in order to realize intrapeptide optimal C13 H-bonds and to give rise to a macrodipole and actually, several experimental researches suggest its absence or marginal presence in solutions.30-32 Instead, its presence have been reported for more longer polyalanine.45 4.7. Energetics. Figure 9 reports relative electronic energies and relative entropies of various conformers with respect to the β-β one, i.e. the energies and entropies of β-β=β-PPII, β-β=PPII-β and β-β=PPII-PPII reactions, as the number of coordinating solvent molecules increases up to twenty-two. For the bare peptide and 2H2O models, the energies of the four conformers spread in about 5 kcal mol-1 and the stability order is: β-β > PPII-β > β-PPII >PPII-PPII. Once four water molecules are considered, the conformers in which the hydrated part adopts the polyproline arrangement (PPII-PPII and PPII-β) undergo a significant stabilization compared to the conformers with the hydrated extended structure (β-β and β-PPII). There is an extra energy gain due to the better coordination of the water molecules in the residue with the PPII conformation. For the PPII-PPII and PPII-β models stabilization progressively increases upon the growing the number of water molecules although it is more pronounced for the conformer PPII-PPII. Even the β-PPII conformer shows a substantial energy gain upon increasing the number of explicit water molecules since the PPII part of the peptide is hydrated. Therefore, the final scale of energy stability is: β-β < β-PPII < PPII-β < PPII-PPII.


27


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Figure 9. Calculated relative electronic energies (∆E) and relative entropies at 298 K (∆S°298) for the Ala3·nH2O (n=2–22) complexes with the peptide in the zwitterion form and in the various unfolded conformations. The Ala3·nH2O energies and entropies with the peptide in the fully extended conformation are taken as references. It is interesting to note that the curves for the β-PPII, PPII-β and PPII-PPII conformers become parallel for models with 18 and 22 water molecules. This is indicative of a reasonable convergence of the relative energy with respect to the number of water molecules. A similar situation has been reported also for the cationic form.26 The peptide structure compactness of PPII residue poses the hydrophilic groups spatially near, thus the models constructed with eighteen number of water molecules are able to express the main hydration effects. However, all 22H2O models have the same number of peptide-water H-bonds, almost the same number of


28

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water-water H-bonds and do not present any significant structural constraint on the water network hence also the hydration convergence should be reached for the β-β conformer. In this context it is useful to note that the three structures (with twenty-two water molecules) optimized for each conformer are close in energy (within few kcal mol-1), hence the relative stability of the four conformers depends slightly on the form of water networks and do not alter final results. Entropy is another important parameter to be considered to describe extended - polyproline II transformations with “hydrated” conformers. Figure 9 reports the entropy variation relative to the β-β conformer obtained for the most stable and homologous nH2O_β-β, nH2O_β-PPII, nH2O_PPII-β and nH2O_PPII-PPII complexes. These data, even though show some fluctuations, point to a certain trend that allow us to draw the following entropy scale for the 22H2O models: β-β > PPII-β ∼ β-PPII > PPII-PPII Figure 9 clearly shows that the stabilization energy of the residues with PPII conformation is counterbalanced by a reduction of entropy. The two components of free Gibbs energy act in the opposite direction thus, the β-β conformation has the highest absolute entropy but the lowest energy stability, while the PPII-PPII has the lowest entropy and highest energy stability. For the mixed β-PPII and PPII-β conformations, an intermediate situation occurs. These findings are in agreement with experimental evidence that showed this molecule mainly adopting the PPII helix at a low temperature,30-32 while on increasing the temperature, the fraction of β-strand quickly grows and becomes predominant. From an experimental point of view, various techniques were used to enquire the equilibrium conformation of residues, however, the UV-CD,46 VCD,47 polarized Raman,48 two-dimensional


29


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IR,49 and 1H-NMR50 spectroscopies turn out to be more promising. The CD spectra provide information on the overall conformational populations of PPII and β-strand independently on the involved residue, thus an overall PPII fraction of 0.84 has been reported (χPPII=2χPPII-PPII + χPPII-β + χβ-PPII).31,32 Conversely, the 1H-NMR measurements provide information for each specific residue, hence the PPII populations around the two peptidic bonds can be evaluated separately.3032

Note that the 1H-NMR experiments give the net PPII population on central and C-terminal

residues [χPPII(central)=χPPII-PPII+χPPII-β and χPPII(C)=χPPII-PPII +χβ-PPII], but they do not allow to evaluate specific amounts of the four conformers present in solution. In other words, 1H-NMR analyses report an independent behavior of the two residues and two values of ∆H°298 and ∆S°298 related to the central and C-terminal residues were derived.30-32 Computed values of ∆H°298, ∆S°298 and ∆G°298 for the zwitterion trialanine 22H2O models in the four peptide conformations are reported in Table 2 along with experimental data derived from CD and 1H-NMR spectroscopies. The table also reports the computed and experimental data of the cationic trialanine for comparison. These data clearly show that a single β→PPII transformation around the central or C-terminal residue implies an enthalpy gain for both zwitterion and cationic trialanine. Also the second β→PPII transformation is exothermic and for both zwitterion and cation the PPII-PPII structures are the most stable. The enthalpic preference of the Ala3 to adopt PPII structure is independent of the protonation state of the peptide in agreement with experimental evidence. In fact, both Toal et al.31 and Oh et al.32 reported the same set of enthalpies for both zwitterion and cation trialanines.


30

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Table 2. Calculated relative enthalpy, entropy and Gibbs free energy at 298 K (∆H°298 and ∆G°298, kcal mol-1, ∆S°298, cal mol-1K-1) for the zwitterion Ala3·22H2O complexes and cationic in the four conformations. The fully extended β-β conformation is taken as a reference. Experimental data are reported for comparison. Ala3H+

Ala3 (zwitterion)

∆H°298

M06-2Xb

PPII-β

β-PPII

PPII-PPII

PPII-β

β-PPII

PPII-PPII

-11.5

-7.8

-13.3

-8.0

-4.9c

-13.3

Graf et al.30

∆S°298

-6

Toal et al.31

-4.9

-2.5

-7.4

-4.9

-2.5

-7.4

Oh et al.32

-3.9

-2.9

-6.8

-3.9

-2.9

-6.8

M06-2X

-18.0

-19.2

-32.9

-16.7

-7.3c

-22.3

Graf et al.30 Toal et al.31

∆G°298

-15 -13.2

-7.7

-20.9

-13.0

-7.1

-20.1

Oh et al.32

-12

-10.7

-22.7

-12

-10.7

-22.7

M06-2X

-6.1

-2.1

-3.5

-3.0

-2.7

-6.7

Graf et al.30

a

a

-1.5

Toal et al.31

-1.0

-0.2

-1.2

-1.0

-0.4

-1.4

Oh et al.32

-0.3

+0.3

0.0

-0.3

0.3

0.0

Computed values for cationic trialanine were taken from references 26 and 27.

b

∆H°298 were computed using the M06-2X/aug-cc-pVTZ/PCM relative electronic energies and the M06-2X/6-31+G*/PCM zero-point energies and thermal corrections to enthalpy. c

These values refer to the 22H2O_β β-PPII_B structure while those previously reported referred to the 22H2O_β β-PPII_C.27

The favorable peptide-water electronic interactions for PPII residue conformation is accompanied by an entropy reduction when a single residue of the β-β conformer transforms in PPII and also the second β→PPII is accompanied by an entropy decrease (Table 2). Nevertheless, in these cases the entropy reduction seems to be more pronounced for the


31


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zwitterion than for the cationic peptide. The entropy reduction in β→PPII transformation is well established and actually 1H-NMR measurements report a two-steps sequence for the β-β → PPIIPPII transition. Nevertheless, Oh et al.32 reported the same set of values for zwitterion and cation forms, while Toal et al.31 reported a slightly more exoentropic values for the zwitterion, in qualitative agreement with present data. Therefore, present computed ∆H°298/∆S°298 set satisfactorily agree with experimental data, even though a quantitative comparison is modest, especially for enthalpies which are about twice the experimental values. Computed and experimental ∆H°298/∆S°298 values can be used to derive Gibbs free energy (Table 2) and also in this case the computations predict qualitatively the correct ordering of conformers stability. However, because of the exothermicity overestimation the computed ∆G°298 are more exoergonic with respect to experimental values. Both the simplified considered model and limitations of the electronic structure method may be responsible for these shortcomings. The latter issue has recently been explored for hydrated cationic trialanine adopting two alternative functionals, i) the hybrid generalized gradientapproximate (hybrid-GGA) functional, B3PW91; and ii) the non-empirical meta-GGA functional, TPSS.27 These alternative electronic structure approaches produce similar results as far as the geometry of models is concerned. However, the peptide-water molecules interaction electronic energies, ∆Eint (Figure 4), are considerably less exoenergetic and also enthalpy variations for the β→PPII are less exothermic, up to the point that, the exothermicity is underestimated with respect to experimental data. These results emphasize the crucial importance of the electronic structure method in the peptide-water interactions evaluation.27 Furthermore, the computation difficulties in evaluating the H-bond strength are well known and


32

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this aspect could be better addressed in the near future when large scale MP2 and CCSD(T) computations will be realizable. In principle, also the bottom-up approach could be improved considering a more elaborate water molecule surrounding as we did for the Ala3⋅41H2O model in the β-β conformation. However, this would be a very lengthy and tedious procedure that could be carried out for small peptides but becomes impractical when the size grows. The simplicity of the proposed method which makes it appealing and easy to apply at tetra and penta-peptides, would be lost. Considering the simplicity of the adopted solvent modeling and the approximations in the theoretical approaches, the experimental/computation comparison for both ∆H and ∆S is satisfactory at the qualitative level. This issue is of great significance since it proves that very complicated phenomena occurring in real solutions can be modeled using a simple and intuitive microhydration scheme, while cohesive forces are evaluated without introducing any empirical parameters.51-54 Even though the proposed procedure is rather long and tedious, because final structures derive from many trials geometry optimizations, it is independent from experimental data and allows us to acquire new scientific and complementary insights in an exclusive computation way. The independence and complementarity are important characteristics for a proper ab initio development. 4.8. The origin of propensity of alanine towards polyproline II helix. There are a lot of studies that try to explain the enthalpic preference of alanine peptides to adopt PPII helix, however, the elementary interactions that actually produce this phenomenon are still missing and are a subject of debate.42,54-60 Both quantum chemical and molecular dynamics recognized the role of explicit water molecules coordination in stabilizing PPII conformation and all these


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studies agree that more efficient H-bonds are formed with respect to β-strand. Furthermore, the better "organized" water molecules in PPII residues, relative to the β ones, involve a higher value of configurational entropy for β conformation.42,54-60 In agreement with these studies, data reported in Figure 9 clearly show a decisive contribution of specific water coordination to the hydrophilic groups of peptide for PPII enthalpy stabilization and entropy destabilization. However, these are the effects not the cause which should be searched in the electronic properties of the bare conformers and peptide-water complexes. Recently, we proposed a new paradigm to rationalize the relation between peptide-water forces and peptide conformation in the β→PPII transformation, the peptidic dipole decoupling.27 The peptidic bond is highly polar (about 5.3 D at M06-2X/6-31+G*/PCM) and, depending on the adopted secondary structure, the dipoles of single residues can couple in different ways. Furthermore, we should consider the local interactions of peptidic dipole and H-bonded water molecule dipoles (2.5 D at M06-2X/6-31+G*/PCM). For a β-strand the peptidic bonds, and thus dipoles, roughly lie on the same plane, but the C=O bonds alternate from one side to the other side of the chain. The dipoles of adjacent residues have opposite orientations, thus they realize the best head-to-tail coupling. The dipole of each residue is largely annihilated and has a low tendency to interact with H-bonded water molecules. Conversely, in the PPII-helix, the dipoles of adjacent residues are rotated by ∼120°, their coupling is only partial therefore, they have a great propensity to interact with the H-bonded water molecules. To prove this qualitative interpretation, quantum chemical analysis on electronic cloud distribution around peptide can clarify some aspects of the nature of the intrapeptide interactions


34

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and the peptide-water interactions. To this purpose remarkably important are the partial atomic charges (presently computed by the modified Hirshfeld CM5 procedure36,37) and the related electrostatic interactions, V in kcal mol-1, evaluated with the formula:

n

V =

∑∑ i =1

qiq j

n j>i

4 πε

0

⋅ ri , j

n

=

n

∑ ∑ 331.906 i =1

j>i

qiq j ri , j

(3)

where n is the number of atoms in the peptide, qi and qj are the partial atomic charges in e.u., and ri,j is the interatomic distance, in Å. For the peptide-water complexes also the interaction energy ∆Eint (eq. 1), which includes all classical and quantum terms, is useful. Given the importance of the intrapeptide interactions in the proposed peptidic dipole decoupling mechanism and also the easiness to make calculations on bare peptides, the CM5 atomic charges and the electrostatic interactions of a large series of peptides (AlaXH+, AlaX (zwitterion), X=3-4 and AcAlaYNH2 Y=2-3) were carried out in the β and PPII conformations (Tables S2 and S3). The cationic and zwitterion trialanines and the protected dialanine, AcAla2NH2, can adopt four structures with residues in the β and PPII conformations, while cationic and zwitterion tetraalanines and the protected trialanine, AcAla3NH2 can adopt eight different conformations. For all the six bare peptides, presently considered, the CM5 atomic charges undergo minor variations as residues change their conformation (Figure S1, and Tables S2 and S3), so there is no significant quantitative difference between the various atoms, rather the spatial distribution of the charges changes.


35


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Page 36 of 50

Table 3. CM5 electrostatic energy, V in kcal mol-1, for bare peptides in all possible β and PPII conformations. β-β

β-PPII

PPII-β

PPII-PPII

Ala3

-393.3

-385.1

-387.0

-379.5

Ala3H+

-437.4

-434.9

-433.1

-431.0

AcAla2NH2

-402.8

-398.6

-398.0

-394.6

β-β-β

β-β-PPII

β-PPII-β

PPII-β-β

β-PPII-PPII

PPII-β-PPII

PPII-PPII-β

PPII-PPII-PPII

Ala4

-500.3

-491.5

-493.6

-495.1

-485.0

-486.4

-488.8

-480.5

Ala4H+

-553.1

-550.5

-548.7

-548.6

-546.3

-546.3

-544.6

-542.2

AcAla3NH2

-517.1

-512.8

-512.8

-511.9

-509.1

-507.3

-508.1

-504.5

Beside the terminal ionized groups, the more significant charge separations are observed for atoms involved in the peptidic bonds with the following average values: O = -0.39, C = 0.30, N = -0.46, H = 0.33 eu. Once these atoms and the related dipole are rotated by about 120° in the β → PPII transformation, the electrostatic interactions, V, computed by equation 3 (Table 3) becomes less negative as qualitative predicted in our peptidic dipole coupling. An average increase of 4.5 kcal mol-1 in V is computed for each β→PPII residue transformation in both non-ionized AcAla2NH2 and AcAla3NH2 peptides. For ionized peptides, similar trends are observed but variations are less pronounced for cations, 3.5 kcal mol-1, and more pronounced for zwitterion, 6.5 kcal mol-1. The marked increase of V for the zwitterion are indicative of a strong coupling between the carboxylic dipole with peptidic groups. These data support our hypothesis that βstrand is stabilized by intrapeptide dipole coupling. The reduction of this intramolecular interaction will give the opportunity to PPII-helix to express better bonds with water molecules and then to be stabilized by intermolecular effects. The electrostatic hydration interaction, ∆Vint, and the quantum chemical hydration energy, ∆Eint, computed as difference between isolated water cluster/bare peptide and their assembly complex


36

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at the peptide-water optimized geometry (eq. 1), can give some insights about the forces involved in solvation phenomena. Data in Table 4 show that electrostatic peptide-water interaction is the main contribution of the overall hydration energy even though its importance decreases a little bit for large solvated peptide. For models with more than twenty-two water molecules, electrostatic interactions are about constant ∼ -80 kcal mol-1 even though quantum chemical ∆Eint becomes more negative. This indicates that other forces, of quantum chemical nature, are still active. Models with two and four water molecules which have hydrated only the β residue (β-β and βPPII) have less negative ∆Vint with respect to conformers with PPII hydrated part (PPII-β and PPII-PPII). This is indicative of more efficient electrostatic intermolecular interactions. Data for more hydrated systems show that peptide-water electrostatic interactions also depend on the surrounding water configuration. In any cases, 18H2O_β β-β β and 22H2O_β β-β β models have the less negative ∆Vint values still emphasizing the weaker propensity of residues with β conformation to interact with water molecules. The importance of electrostatic interactions in peptide-water formations was also assessed by Scheiner et al..61 They reported a series of DFT studies to analyze how and why dipeptide-water H-bonds vary with the internal conformation of the protected HCOGlyNH2 peptide. They noted that the peptide >NH group reduces its proton donor capability when the residue assumes an extended β conformation. Among the possible considered reasons, they ascribe the >NH⋅⋅⋅OH2 strength reduction in β conformation to an electrostatic origin. They assert that the hydrogen bond in the β conformer is weakened by the coupling of the O atom, of the adjacent peptide group, to the NH proton donor. The partial negative charge of this O atom strongly perturbs the


37


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Page 38 of 50

electrostatic potential that is experienced by an incoming proton acceptor in full agreement with our findings. Table 4. CM5 electrostatic (∆Vint) and quantum chemical (∆Eint) hydration energies (kcal mol-1) for the zwitterion Ala3⋅nH2O models in the four unfolded conformations. 2H2O_β-β

∆Vint -13.6

∆Eint -14.3

18H2O_β-PPII_A'

∆Vint -82.6

∆Eint -87.1

2H2O_β-PPII

-13.2

-14.3

18H2O_PPII-β

-80.4

-90.4

2H2O_PPII-β

-16.2

-13.8

18H2O_PPII-PPII_A

-78.3

-97.4

2H2O_PPII-PPII

-14.2

-13.7

18H2O_PPII-PPII_B

-76.6

-92.6

4H2O_β-β

-25.1

-26.5

22H2O_β-β_A

-77.9

-87.4

4H2O_β-PPII

-24.1

-26.5

22H2O_β-β_B

-77.2

-89.2

4H2O_PPII-β

-33.7

-29.8

22H2O_β-β_C

-73.4

-89.2

4H2O_PPII-PPII

-30.5

-28.8

22H2O_β-PPII_A

-82.1

-91.1

9H2O_β-β_A

-42.8

-48.0

22H2O_β-PPII_A'

-79.6

-90.2

9H2O_β-PPII

-38.7

-44.5

22H2O_β-PPII_B

-81.9

-93.4

9H2O_PPII-β

-54.7

-55.7

22H2O_PPII-β_A

-78.9

-95.6

9H2O_PPII-PPII

-58.9

-58.8

22H2O_PPII-β_B

-74.1

-94.1

15H2O_β-β

-66.9

-71.9

22H2O_PPII-β_C

-75.5

-91.1

15H2O_β-PPII_A

-64.9

-72.9

22H2O_PPII-PPII_A

-82.7

-97.2

15H2O_β-PPII_B

-69.3

-72.0

22H2O_PPII-PPII_A'

-80.8

-101.3

15H2O_PPII-β_A

-65.8

-75.6

22H2O_PPII-PPII_B

-82.1

-94.3

15H2O_PPII-β_B

-66.9

-78.5

24H2O_β-β

-76.6

-92.4

15H2O_PPII-PPII_A

-77.7

-89.3

27H2O_β-β

-80.1

-96.7

15H2O_PPII-PPII_B

-77.7

-82.9

32H2O_β-β

-79.4

-98.9

18H2O_β-β

-73.5

-80.0

38H2O_β-β

-78.4

-101.4

18H2O_β-PPII_A

-73.7

-87.4

41H2O_β-β

-76.4

-102.3


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The crucial role of environmental effects on PPII-helix has been also documented for a large series of AcAlanNH2 (n up to eleven)44,62 and specifically PPII-helix does not exist as a minimum in the Born-Opphenimer surface derived from quantum chemical calculations in the gas phase, while all possible conformations are found simply considering nonspecific hydration effects, i.e. the coupling of peptidic dipoles with a continuum medium. 5. CONCLUSIONS Microstructural water/peptide interface stepwise construction, carried out for the zwitterion trialanine, allows us to evaluate the effects of the very first hydration shell on its molecular properties. Even though the proposed cluster approach is a challenge, since it is not automated and many trials need to be carried out to get reliable structures, the good quality of present results along with those reported for cation trialanine,26,27 protected alanine AcAlaNH225 and GXGH+,63 suggest that the procedure is quite robust and accurate. It provides a means for thermodynamic function evaluation of small peptide in solution without introducing any empirical external parameters, hence it has two important features i.e. independent results and complementary with respect to experimental data that make it appealing for further explorations. Present data reveal that peptide-water hydrophilic interactions are of primary importance and their rationalization allow us to understand factors that govern hydration phenomena. It has been shown that the enthalpic preference of PPII-helix over the extended β, due to water molecules coordination, originates from a competitive intrapeptide or intermolecular electrostatic stabilization of highly polar atoms of peptidic bonds.


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Page 40 of 50

SUPPORTING INFORMATION Additional optimized structures are in Figures S1 and S2, dihedral angles are in Table S1 and CM5 atomic charges are in Tables S2 and S3. A complete list of Cartesian coordinates of all structures presently analyzed are also reported. This material is available free of charge via the Internet at http://pubs.acs.org.” AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] ACKNOWLEDGMENT AND DEDICATION This work was supported by the Ministero dell’Istruzione, dell’Università e della Ricerca (MIUR) (PRIN 2010-2011, 20109Z2XRJ_003) and Università di Catania within the FIR project. This article is dedicated to the memory of Carmela Spatafora, our colleague and friend.

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Table of Contents Graphic


50

Effects of Hydration on the Zwitterion Trialanine Conformation by

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