Chapter 4. Glutamic Acid in Solution - Correlations

Transcription

1 Chapter 4 Glutamic Acid in Solution - Correlations 4. Introduction Glutamic acid crystallises from aqueous solution, therefore the study of these molecules in an aqueous environment is necessary to understand how the molecules behave in such a solvent - how does the water influence the amino acid and vice versa? Correlation between two atoms can be described using the RDF (see Chapter ), which shows the probability of locating an atom at a distance r from the atom of interest. Studying the RDFs between glutamic acid and the surrounding water will provide information on the nature and strength of these interactions. RDFs give information on the location of the water molecules around glutamic acid, providing an indication of which functional group has more affinity with water. The coordination numbers provide an indication of the number of oxygen atoms from water molecules, and, by extension, water molecules, around the different glutamic acid atoms. The first system studied consists of a single molecule of glutamic acid immersed in water. The interactions of the functional groups of glutamic acid with water have been analysed via the RDF of the solute atoms with the water atoms. The different isoelectric forms discussed previously were also investigated in a single solute molecule system. In order to compare the water structure around a glutamic acid molecule and around a molecule from the two crystalline polymorphs of glutamic acid, a single α and a single β molecule extracted from the respective crystals were also studied, by keeping them in a fixed conformation. The strength of the solvation layer around all the single glutamic acid molecules was investigated via means of hydrogen bonding in that shell. Finally, systems representing a supersaturated solution were studied, providing information on the glutamic acid water interactions. 4. Results and discussion The pair distribution functions g is calculated at every timestep and the results are based on the simulations described in Chapter 3. 78

2 4.. Single Glutamic Acid Molecule A single glutamic acid molecule is expected to perturb the surrounding water structure only locally. The RDF of the water-water interactions for both pure water and the solution containing a single glutamic acid molecule gives an indication of that perturbation (Figure 4.). The RDFs for OW-OW, OW-HW and HW-HW in the solute system are very similar to those in the RDFs for pure water. The peaks are in the same places and approximately the same shape. However, the intensity of the first peak in each RDF is lower than that for pure water, indicating a very small perturbation caused by a single molecule of glutamic acid. The water-water RDF for the glutamic acid in water system is in good agreement with the work of Campo on glycine in water (Campo, 6). Glycine is the smallest of all the amino acid molecules and with a hydrogen residue. The maxima and minima of the water-water RDF for a single glycine molecule in aqueous solution correspond to those for system containing a single molecule of glutamic acid (see the supporting material of Campo (6)). The direct interactions between glutamic acid and SPC/E water, including the nature of the solvation shell can be illustrated by the RDFs for atoms in glutamic acid to those in water. The focus will be on the interactions between the atoms in water and those in the functional groups of glutamic acid (carboxylic groups and amine group) as these are likely to be the strongest. The hydrogen bonds in water are represented by the RDF of the OW-OW and OW-HW pairs. Because a hydrogen bond is formed by a hydrogen atom between two oxygen atoms, the first peak of the OW-HW pair correlation function is at a shorter distance than the OW-OW first peak by Å, the length of the OW-HW bond. The second peak of the OW-HW is made by the second hydrogen atom of the same water molecule involved in the first RDF peak. This trait can be used in other pair correlation functions to identify a hydrogen bond between a solute and water. 79

3 solution pure water g HWHW g OWHW 4 g OWOW Figure 4.: RDF of the water atoms in a pure water system (dotted line black) and in a solution with one glutamic acid in water (plain line) Figure 4. shows how the carboxylic ends of the glutamic acid zwitterion (O, O and OH) interact with the neighbouring water molecules compared to the bulk water interactions. The correlaion between the carbonyl oxygen atoms of glutamic acid (O and O) and the water atoms (OW and HW) are very similar to those for bulk water, which suggests that the water is strongly bound to the carbonyl oxygen atoms. In particular, there appears to be a significant structuring effect for water around the carbonyl oxygen of the protonated carboxylic acid (O) atom of glutamic acid, as evidenced by the sharp first peak in the O-HW RDF and the enhanced first minimum in the O-OW RDF compared to that for pure water. This indicates a hydrogen bond interaction, where the water molecule is held more firmly in place by the O atom in glutamic acid than it would be by another water molecule. The correlation between oxygen atoms in the deprotonated carboxylic group (O) and water appears to be very similar to that for bulk water, while the correlation between the oxygen on the hydroxyl group of the protonated carboxylic group (OH) and water appears to be weaker than for bulk water, which may be an effect caused by the interaction between water and the neighbouring O atom. The coordination numbers in Table 4. show that O and O 8

4 atoms are surrounded by, on average,.6 and. water molecules, respectively. While the peak positions for O-OW and O-OW correlations are the same as OW-OW correlation, the heights are reduced in the glutamic acid cases. This could be due to flexibility in the solvation cage or to the conformation of the glutamic acid. For the case of the hydroxyl oxygen (OH), the peak heights are reduced compared to the bulk water, although they are at the same distances; the intensity of the first peak is substantially lower than for the water-water interactions, but broader in the case of OH-HW interaction: the peak width at half height is.34 for g O-OW and g O-OW, compared to.48 for g OH-OW. This suggests less ordering between the OH oxygen atoms and the surrounding water. The coordination numbers from these RDFs are. for OH-OW and 4.4 for OH-HW, which is higher than for the carbonyl oxygens. This indicates that the water molecules involved in hydrogen bonds with the hydroxyl oxygen are either involved in the solvation of the hydroxyl hydrogen (HO) atom, co-ordinated with other water molecules that are involved directly in the solvation of the OH atom, or both. 8

5 4 g OOW g OWOW g OHW g OWHW g OOW g OWOW g OHW g OWHW g OHOW g OWOW g OHHW g OWHW Figure 4.: RDFs for glutamic acid oxygen compared with water-water RDFs. g N3OW g OWOW g N3HW g OWHW g HOW g OWHW g HHW g HWHW r(å) r(å) Figure 4.3: RDFs for the amine group of glutamic acid compared to water-water RDFs. 8

6 The other functional group of interest in glutamic acid is the amine group formed by the N3 and three H atoms. The correlations between the amine group and the oxygen from water show that the water molecules are structured around the amine group (see Figure 4.3). The g H-OW shows a similar pattern to g OW-HW, and g N3-OW is very close to g OW-OW, suggesting hydrogen bonds between the amine group and the surrounding water. In the first hydration shell the distance between N3 and OW is slightly larger than the OW-OW distance, due to the nitrogen having a larger atomic radius than oxygen. The first peak in the RDFs for N3-OW and OW-OW lies at distances between the first two peaks of H-OW and OW-HW, respectively, which is characteristic of a hydrogen-bonded system. The first peak in the RDF of the N3-HW pair is at 3.5Å, compared to.8å for the RDF for the OW-HW pair. The nitrogen atom is already covalently bonded to three hydrogen atoms as well as the bulk of the glutamic acid molecule, and this sterically hinders direct interaction between the amine nitrogen and a water hydrogen atom. The RDF for H-OW presents lower and slightly shifted peaks compared to OW-HW, which means that the hydrogen atoms from the amine group interact less with the water atoms compared to pure water. This is due to the fact that these hydrogen atoms belong to a charged functional group, making it harder for these atoms to form hydrogen bonds with the surrounding water molecules. The N3-HW RDF presents a first peak shifted compared to OW-HW, which denotes larger distances between N3 and HW. The water molecules surrounding the amine group will be orientated so that their hydrogen atoms are facing away from the glutamic acid molecule towards the bulk water (Figure 4.4), hence shifting the first peak of N3-HW RDF. The coordination numbers for the amine group confirm these interactions with 3.8 water molecules around the nitrogen atom, and.9 around each of the hydrogen (Table 4.). The coordination numbers for a single glutamic acid molecule in water are close to those for a single glycine molecule in water (.67 and.94 for the atom pairs O-OW and H-OW, respectively) (Campo, 6), for an alanine molecule (.3 and.9 for O-OW and H-OW, respectively) (Degtyarenko et al., 7), and for aspartic acid (.7 for O-OW and around 3 for N3-OW) (Kim and Jhon, 994). There are small differences between the amino acids, especially around the carboxylic group, that are due to the difference of methodologies and force fields, as Raman spectroscopy performed on various amino acids at neutral ph suggested that the side chain affects the structure of water very little (Ide et al., 997). 83

7 Figure 4.4: the water molecules around the amine group are orientated with the hydrogen atoms facing the water bulk RDF n i r min (Å) g OW-OW (pure water) g OW-HW (pure water).9.43 g HW-HW (pure water) g OW-OW (solution) g OW-HW (solution).9.43 g O-OW g O-HW.6.58 g O-OW g O-HW..33 g OH-OW. 3.3 g OH-HW g N3-OW g N3-HW g H-OW.9.53 Table 4.: coordination numbers for water-glutamic acid RDFs 84

8 4... Effect of Temperature 4 g OWOW g OOW g OWHW g N3OW g OOW g HOW K K Figure 4.5: effect of temperature on the solution and on the interactions of water with glutamic acid at 6K (dotted line) and 3K (plain line) The effect of temperature on the interactions of glutamic acid molecules with the surrounding water was also investigated. The RDFs for the simulation run at 6 K shows very little difference to those obtained from the simulations at 3 K. Figure 4.5 shows that the peaks of the glutamic acid-water RDFs are higher and sharper at the lower temperature, which indicates that the water around the glutamic acid becomes slightly more structured as the temperature decreases, as suggested by Bowron et al. (). 4.. Isoelectric Forms The variations in the conformations of the different isoelectric forms are relatively small. These protonations and deprotonations will probably have a greater effect on the interactions between the functional groups and water, which can be analysed by the relevant RDFs. 85

9 g O-OW g O-HW g O-OW g O-HW g OH-OW g OH-HW zwitterion Figure 4.6: RDFs of the interaction of carboxylic group with water. The bottom two graphs only display two curves each due to the absence of the OH atom in the - and - forms (see Figure 3.). Figure 4.6 shows that the RDF of O-water atoms is similar for all isoelectric forms, which means that the deprotonated oxygen from the - and - forms behaves like the other O oxygen in the zwitterion. The peaks are sharper for the two negatively charged species compared to the zwitterion and the + form, denoting a strong hydrogen bonding with the water molecules because both oxygen atoms on C are deprotonated, leaving more space for the water molecules to interact with the carboxylic group. Figure 4.7 illustrates the kind of hydrogen bonds that occur during the simulation between the carboxylic groups of the - form of glutamic acid. 86

10 Figure 4.7: Two examples of hydrogen bonding around the carboxylic groups for the - form. The water molecules not involved in a hydrogen bonding with O or O have been removed for clarity. Figure 4.8: Two examples of hydrogen bonding around the carboxylic groups for the - form. For the other carboxylic group (O), the only form that has higher peaks in the RDF than the zwitterion is the - form, due to the deprotonation of the nearby amine group, decreasing the steric hindrance around the carboxylic group on C. The - form shows a 87

11 decrease in the first peak of both the O-OW and O-HW RDFs, which can only be explained by the competition of the other carboxyl group (Figure 4.8). In the - form, both carboxylic groups carry two carbonyl oxygen atoms each (two O on C and two O on C), and their respective correlation functions, i.e. O-OW and O-OW show that the water molecules prefers to interact with O (the first peak intensity is around 4 and for O-OW and O-OW, respectively). So it seems that in the case of the - form, the deprotonation of the carboxylic group on C affects the interaction of the C carboxylic group with the water molecules. Because of the proximity of the C carboxylic group with the amine group, the water molecules preferably hydrogen bond to the other, less encumbered group, at the other end of the molecule on C. The O- OW and O-OW RDF for the - form have a first peak intensity of.5 for both, which means that once the amine group is deprotonated as well as the carboxylic groups, the interactions of water with the both carboxylic groups are equivalent in intensity. The + form shows a different pattern for the O-OW RDF: the first peak is lower, shifted and a lot broader than all the other forms denoting a strong interaction with the water, but with less structure around the only O of the molecule. The proximity of the amine group induces less structure with the water around the O atom compared to the O atom. The OH atom is only present in the zwitterion (on C) and in the + form (on C and C). The corresponding RDFs show that the hydroxyl oxygen atoms of both forms present the same interaction with water, independently of which end of the molecule they are located. The peaks are slightly higher for the zwitterion than for the + form, due to the steric hindrance of the amine group close to OH in the + form, but the effect is smoothed by the average over the two OH atoms present in that particular state of glutamic acid. The structure in Figure 4.9 shows that when the carboxylic groups are protonated they form equivalent interactions with the surrounding water molecules. 88

12 Figure 4.9: + form of glutamic acid and the water molecules around the carboxylic groups. g N3-OW g N3-HW g H-OW g H-HW zwitterion Figure 4.: RDFs of the interactions of the amine group with water in the different isoelectric forms of water. 89

13 The amine group, on the other hand, shows more divergence between the different isoelectric forms (Figure 4.). The RDFs formed by the N3-OW and H-OW pairs for the + form of glutamic acid show higher peaks than for the zwitterion. The OH atoms present in the + form is the only difference compared to the zwitterion. It was show in the previous chapter through the analysis of the conformation that the OH atom was located further form N3 compared to O. The influence on the interaction between N3 and the water atoms arise from the location of the hydroxyl group by increasing the correlation with water. The - form, which carries an NH group rather than the NH 3 + group common to the other forms, loses the attraction to the water molecules altogether, and shows no evidence of hydrogen bonding or solvent structure around that group α and β Molecules in Water In order to investigate the solvation shell around the molecules that are found in the crystals, a single molecule was extracted from the α and β crystal structures and was simulated in SPC/E water. In each case, the glutamic acid molecule was kept in a fixed conformation at a fixed position, but the water molecules were allowed to move. The resulting correlation functions of the two conformations in water were compared to the interactions from the single glutamic acid molecule in water simulation (Figure 4. and Figure 4.3). 9

14 g OH - OW g OH - HW g O - OW g O - HW 3 4 g O - OW g O - HW α frozen β frozen not frozen Figure 4.: RDF of O, O and OH atoms with water around an α and β molecule, compared to a glutamic acid molecule that is allowed to move. In general, the interaction with water around the α molecules seems to be slightly more significant than for the β molecule as the first peak of the RDFs formed by the glutamic acid atoms with the water atoms are higher (Figure 4. to Figure 4.3). This is particularly true around N3, OH and C3. It seems that the α conformation favours the formation of hydrogen bonds with the water molecules that surround it compared to the β conformation. However, it was shown in Chapter 3 that a flexible glutamic acid molecule is significantly more β-like than α-like in terms of conformation. The correlation functions for a glutamic acid molecule that is allowed to move are closer to those of the β molecule for O and OH, and for the backbone carbon atoms (Figure 4. and Figure 4.3). It is only around the H of the amine group that the interactions with water for an unfrozen solute are similar to those around the α molecule (Figure 4.), which suggests that the H-OW interactions are less important in the determination of the glutamic acid conformation. 9

15 g N3 - OW g N3 - HW g H-OW g H-HW α frozen β frozen not frozen Figure 4.: RDF of N3 and H with water around an α and β molecule, compared to a glutamic acid molecule that is allowed to move. g C3 - OW g C5 - OW g C - OW g C - OW g C4 - OW g HP - OW α frozen β frozen not frozen Figure 4.3: RDF of the backbone carbon atoms (C, C, C4, C4 and C5) and HP with water around an α and β molecule, compared to a glutamic acid molecule that is allowed to move. 9

16 Of all the atoms in glutamic acid involved in the torsion angles that have been calculated previously (Chapter 3), only C, N3 and C are involved in only one torsion angle each. The interaction between these particular atoms and water therefore can provide an indication of the effect of water on the conformation of the molecule. N3, included in T, presents a correlation function with water that is slightly closer to that of the β polymorph. g C-OW shares the same first peak as both polymorphs, but presents a second peak closer to that of β. C, involved in only one torsion angle (T3) presents a correlation function with water very close to that of both polymorphs. One can note that the C5-OW correlation for an unfrozen solute is close to a β molecule. Because a torsion angle is made by four distinct atoms, studying their respective distribution functions does not provide a clear relationship between the interactions with water and the conformation of the molecule. What can be concluded from this analysis is that there is a general tendency for the atoms of a fully flexible glutamic acid molecule involved in the torsion angles monitored to favour correlation with water that is close to the correlation found around the atoms of a frozen β conformation Solvation Shell around a Single Molecule in Water The structure of the immediate solvation shell around a single molecule of glutamic acid was investigated for all the isoelectric forms, as well as for the fixed α and β conformations. Figure 4.4 illustrates the solvation shell of the zwitterion form. The solvation shell was defined as all the water molecules located within a radius of 3.8Å of the glutamic acid molecule. That cut-off distance has been chosen as the average first minimum of the pair correlation between the glutamic acid backbone atoms, namely N, C and O, and the water atom OW. The number of water molecules in the solvation shell will then depend on the conformation of the glutamic acid molecule. The average number of hydrogen bonds that a water molecule from the solvation shell shares with the rest of the water was calculated and averaged over the simulation. The number of hydrogen bonds formed between the water molecules of the solvation shell with other molecules within the solvation cage is an indication of the strength of the internal shell and has also been calculated. The results of these calculations are presented in Table 4. with their respective standard deviations. 93

17 Figure 4.4: The solvation shell around a glutamic acid molecule Solute molecule Number of water molecules in the solvation shell σ a Number of H- bonds per water molecule in the solvation shell σ a Number of H- bonds per water molecule with other water molecules in the solvation shell zwitterion form form form α β Table 4.: molecules and hydrogen bonding of the solvation shell ( a σ = standard deviation) σ a The water arrangement around the unfrozen zwitterion, - and - forms have the highest number of hydrogen bonds per water molecule compared to the other conformations and 94

18 forms (around 3.7). The water molecules tend to maximise the number of hydrogen bonds, which makes the solvation cage around these molecules the most stable. However, the number of water molecules in the solvation shell around a free moving glutamic acid molecule is closer to the number found around a fixed α conformation than a fixed β conformation. So it appears that the water structure around the solute molecule prefers to be similar to that around a fixed α conformation. The number of hydrogen bonds formed by the solvation shell with other water molecules again confirms the fact that the water arrangement prefers the α molecule to β. The standard deviations (σ) are quite high, around % for most cases, which indicates a movement of the water molecules around the solute molecule along with the formation and breaking of hydrogen bonds. The + form and the frozen beta conformation both have the solvation shell with the lowest number of hydrogen bonds. The conformation of these molecules does not favour a strong hydrogen bond network within the neighbouring water molecules. The number of hydrogen bonds between molecules within the solvation shell follows the same trend as the number of hydrogen bonds between the solvation shell and the rest of the water, with.73 for the zwitterion and.74 for the fixed α molecule. It seems that there may be a contradiction between the glutamic acid conformation (see Chapter 3) and the glutamic acid water interactions (Figure 4. to Figure 4.3 ), where the former prefers the conformation found in the β crystal, while the latter (Table 4.) prefers the conformation found in the α crystal. So does this suggest that it is the solvent that drives initial crystallisation to be in the form of the α polymorph, even though the solute clearly prefers the more thermodynamically stable β polymorph? This begs the question, what needs to happen to make the solute change its conformation? A force field capable of representing each of the two potential energy minima corresponding to the α and β conformations (Davey et al., 997) would allow the glutamic acid molecule to take up these conformations and also more intermediate conformations. The effect of other glutamic acid molecules in the solution might also influence the conformation of the solute molecules. Section 4..5 on supersaturated solutions is the first step in attempting to answer this question. Perhaps a different force field would provide with different conformation. 95

19 The other isoelectric forms display a different solvation shell: the number of water molecules present in the cage is higher than for the zwitterion, but the number of hydrogen bonds formed by it is different between the charged forms: for - and -, the solvation cage is strongly bound by hydrogen bonds, more than for the zwitterion. For the positively charged + form, the number of hydrogen bond drops from 3.7 for the zwitterion to 3.34, indicating a weaker water structure around the + state of glutamic acid. The + form and the frozen beta conformation both have the solvation shell with the lowest number of hydrogen bonds. The conformation of these molecules does not favour a strong hydrogen bond network within the neighbouring water molecules, resulting in a less structured and less stable solvation shell Supersaturated Solution The study of supersaturated systems not only provides information on the structure of the glutamic acid molecules, but also on the structure of the solvent and the nature of the interactions between the solute and solvent molecules. The effect of solute concentration on the bulk water structure is shown in Figure 4.5 for simulations performed in the NVT ensemble. The water-water correlations of solutions of different concentration are very similar, denoting a small effect of concentration on the bulk water structure. However, the perturbation of the bulk water structure does not appear to be a function of concentration. The water-water RDFs appear to be similar for pure water, for 5 and 4 glutamic acids, while those for and glutamic acids are also similar to one another. The hypothesis that this is due to the clustering behaviour of glutamic acid in supersaturated solutions is discussed in Chapter 5. The analysis of the coordination numbers for the water-water interactions of these systems seems to confirm this for the and glutamic acids in water, as they both have a coordination number of 4.4 (Table 4.3). For the 5 glutamic acid system, the coordination number for the OW-OW interactions is 5., compared to 4. for a pure water system (Table 4.3). This shows that the water arrangement in the 5 glutamic acid system is more compact than in pure water; this could be due to individual solvation of the glutamic acid molecules, forcing the water molecules closer. Because of the low concentration of that particular system, the five solute molecules are solvated individually, resulting in the water molecules being brought closer together and consequently increasing the waterwater interactions. This is confirmed by solute-solute interactions of that system, which shows that the glutamic acid molecules are not close to each other for that concentration (see Chapter 5). The coordination number for the 4 glutamic acid system is lower than 96

20 for pure water at 3.4, which indicates that the high level of supersaturation decreases the coordination between the water molecules. This can be explained by the presence of more solute molecules in the solution that prevent some of the water-water interactions to occurs, which are replaced by solvent-solute interactions. 4 g OW - OW g OH - HW g HW - HW r(å) pure water 5 4 Figure 4.5: effect of concentration on the water-water interactions. The abscissa scale has been reduced for clarity. RDF g OW-OW S n i r min (Å) pure water glutamic acid glutamic acids glutamic acids glutamic acids Table 4.3: coordination numbers of g OW-OW for different aqueous solutions. S is the supersaturation with respect to α. The analysis of the glutamic acid-water interactions in the system describes the level of correlation with the water as a function of the concentration. Figure 4.6 describes the 97

21 interaction between the oxygen and nitrogen atoms of glutamic acid (O, O, OH and N3) and the water oxygen atom (OW) for the three concentrations. The solute-solvent interactions for the less concentrated system containing 5 glutamic acid molecules (Figure 4.6) present the same pattern as for a single solvated glutamic acid molecule (see Figure 4.3 and Figure 4.5). The same thing is observed for the more concentrated system containing glutamic acid molecules. The more concentrated system containing 4 glutamic acid molecules presents almost the same interactions with water as for less concentrated systems, with a small decrease in the intensity of the first peak. The increase in concentration does not affect the interactions between the functional groups of glutamic acid and the water molecules. A similar simulation was performed by Campo on glycine at two concentrations; the first system contained glycine molecule in 33 water molecules and the second contained 3 glycine molecules in 3 water molecules. The results showed a small decrease in the interactions between glycine and water due to the packing effect of solvent molecules. The hydration structure is more compact when the concentration is increased compared to infinite dilution (Campo, 6). Another simulation on a more concentrated system containing 5, and 3 tertiary butanol in 35, 78 and 68 water molecules, respectively, revealed no change in the solute-water interactions and the same coordination number was obtained for the first hydration shell. It should be noted however, that even at the smallest concentration, the tertiary butanol molecule is not completely encapsulated by the water (Bowron et al., 998). Tertiary butanol is a lot smaller than glutamic acid, and the concentration used was a lot higher than the ones used here. 98

22 g O - OW g OH - OW g O - OW g N3 - OW Figure 4.6: water-glutamic acid interactions for a system containing 5 (plain gray line), (plain black line) and 4 (dashed red line) glutamic acids in water for O, O, OH and N3 compared to infinite dilution (dotted blue line) The interactions with water for the - form of glutamic acid in the most concentrated system with 4 molecules are presented in Figure 4.7. Only the interaction between O and water are different for the - form compared to the zwitterion, due to the deprotonation of OH. It demonstrates that the - form interacts with water in the same way as the zwitterion for a concentrated system, which was not the case for a single molecule in water (see Figure 4.6 and Figure 4.). The infinite dilution of the - form indicated strong O-OW interactions and weak O-OW interactions (Figure 4.6), while the most concentrated system appears to exhibit similar interactions for O-OW and O-OW, indicating that the water is bound to the oxygen atoms in a similar way. The capacity of water molecules to selectively bind to glutamic acid oxygen sites is dampened by the presence of other glutamic acid molecules in the solution. The interaction of the water atoms with the amine group was similar for the - form and for the zwitterion at infinite dilution (Figure 4.), and remains similar for a concentrated system (Figure 4.7). The O-OW and O-OW are similar to the O-OW interaction 99

23 found by McLain et al. (6), but they found the N3-OW interaction much stronger (see supplementary material of McLain et al. (6)). When the number of solute molecules is high enough, the - form of glutamic acid exhibits interactions between its functional groups and water similar to those of the zwitterion molecules. g O - OW g O - OW g N3 - OW zwitterion - form Figure 4.7: Glutamic acid water interaction for 4 glutamic acid molecules in water; zwitterion (plain black line) and the - form (dashed red line) for O, O and N Conclusion The structure of the water molecules around glutamic acid was determined for a single molecule of the amino acid, and the interactions between the functional groups and the surrounding water were compared with each of the different isoelectric forms of glutamic acid. A strong correlation between the carbonyl oxygen atoms and the nitrogen atom with the water molecules was observed. The conformation of a fixed α molecule favours the formation of hydrogen bonds with water compared to a fixed β molecule. The solvation shell around a fixed α conformation is similar to that found around a fully flexible glutamic acid molecule in water, while the conformation of the solute molecule itself is closer to the conformation found in the β crystal. This could suggest that the solvent plays a crucial role in determining the polymorph that is initially formed. The study of the different isoelectric forms of glutamic acid showed that deprotonation of both the amine group and the carboxylic group on C leads to a rigidity of the amino

24 acid tail. The protonation of the carboxylic group on C leads to a preferred conformation where the hydroxyl oxygen is close to the amine group, as it influences the correlation between the nitrogen atom and water atoms. The AMBER force field is able to reproduce the inter- and intra-molecular structure, with a good agreement between MD and experiments, and shows similar level of ordering (Blieck et al., 5). The conformational study in Chapter 3, on the other hand, suggests that this force field does not correctly predict the two potential energy minima that correspond to the α and β conformations, and is biased towards β. The study of the solute-solute interactions should also give an indication as to the validity of the force field employed here. In Chapter 5, the solute-solute interactions in supersaturated solutions will be investigated as a function of the concentration, in order to determine the structure of potential clusters, and the frequency at which the clusters are forming. The agglomeration of glutamic acid molecules will be examined by looking at the structure and dynamics of the clusters and conformations of the molecules within them.