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A Molecular Dynamics Investigation on the Occurrence of Helices in Polygalacturonic Acid
Bruno Manunza 1*, S. Deiana 1, M. Pintore 1, C. Gessa2
Keywords: polygalacturonic acid, polysaccharides, molecular dynamics
Partially esterified polygalacturonic acid is the main component of pectin in higher plants. It constitutes the mucilaginous soil-root interface and acts as an accumulation phase for nutrients being an important media for the diffusion of ions towards the root absorbing cells. The carboxylic groups and their methyl esters markedly affect the ability of the pectin molecules to bind oppositely charged ions and to form gels. Molecular Dynamics was employed to investigate the conformational equilibrium and the intermolecular interactions of a system constituted by 2 polygalacturonic acid chains, each formed by 24 units of galacturonic acid. The results suggest that, as evidenced for other polysaccharides, a helix based structure could be proposed for the polygalacturonic acid chains.
Acid sugars play an important role in the biochemical processes involved in plant nutrition. They are found, both in monomeric and polymeric forms, on the root surfaces and cell walls. The polymers are the main constituents of the mucilaginous soil-root interface (mucigel): they behave as an accumulator for the nutrients and are involved in the diffusion process of the ions towards the absorbing cells [1-3]. These properties may be due to the polygalacturonic acid (PGA) chains (Fig. 1), which are the main constituents of the root mucilage [4-6]. Electron Microscopy studies provide evidence that these polymers are organised in a fibrillar structure [7-11]. These structures act as selective filters for the nutritive elements and regulate the movement of ions through and out the cells [12].
Fig.1 Polygalacturonic acid (PGA).
The knowledge of only the primary structure of complex carbohydrates is no longer sufficient to understand and explain their function and specificity [13]. The three-dimensional structures of pectin and polyuronic acid determine their interactions with other ions, molecules and macromolecules and are significant for their function and biological activity [14-16].
In previous work [17], we applied Molecular Dynamics to the study of the motion of Ca2+ ions around four units PGA chains, and found evidence for the existence of channels where the Ca2+ ions preferentially move. Computational chemistry methods may greatly help in the determination of the three-dimensional structure of polysaccharides allowing to understand and explain the behaviour of the PGA chains and the diffusion of ions inside. This study reports the results of a Molecular Dynamics survey about the aggregation process between PGA chains counting 24 galacturonic acid monomers. The evidence of helix formation among the chains is reported. The driving force of the aggregation process is the formation of interchain hydrogen bonds.
The length of the PGA chains on which the Molecular Dynamics (MD) experiments were performed was 24 units, with an overall molecular weight of 4264 per chain. The conformation of the chain used as input in the MD experiment was previously optimised via a Molecular Mechanics (MM) calculation and is shown in Fig. 2.
Figure 2. The MM optimized conformation of the 24 units PGA chain. Click inside the figure to retrieve the pdb file.
The DLPOLY2 [18] program was used to carry out the Molecular Dynamics (MD) experiments. We used the AMBER plus GLYCAM force field [19]. The partial atomic charges were calculated by fitting the electrostatic potential computed by ab initio HF-SCF calculations at the 6-31G*accuracy level. The GAMESS program [20] was employed to perform both the ab initio and charge fitting computations. The values of the fitted charges are shown in Fig.1. A relative dielectric constant value of 1.0 and a spherical cut off of 20 Å for coulombic and long range forces were adopted in all the simulations.
Several MD 1000 ps trajectories were performed on the systems with 1, 2, and 3 PGA chains. The runs were stopped after 1000 ps as no significant variation was observed in the total energy during the last 300 ps.
A snapshot of an equilibrated conformation is shown in Fig. 3. The radial distribution functions (g(r)) between the various types of oxygen atoms in the PGA chain are shown in Fig. 4.
Figure 3. An equilbrium conformations extracted from the trajectrory computed for the 1 chain system. . Click inside the figure to retrieve the pdb file.
The peaks at r»3Å in Fig. 4 are attributable to intramolecular hydrogen bonding between vicinal hydroxyl groups and between the endocyclic oxygen and the nearest hydroxyl group. The remaining peaks are due to the periodic structure of the PGA.
Figure 4. Radial Distribution Functions computed for the 1 chain system between the oxygen atoms of the PGA. OH: Oxydril Oxygens, OA: sp3 Oxygens in the COOH groups, OS: endocyclic Oxygen, and OG: glycosidic Oxygen.
The analysis of the MD trajectory shows that the two chains strongly interact reciprocally by the formation of hydrogen bonds which involves both the carboxylic and the hydroxyl functions in the PGA chain. The g(r) between the Oxygen and Hydrogen atoms are shown in Fig. 5.
Figure 5. Radial Distribution Functions between the hydroxyl (OH) and the carboxyl (O2) oxygen atoms and the hydroxyl (HO) hydrogen atoms in the PGA chains.
The pronounced peaks at r»2Å angstrom are representative of hydrogen bonds formation among the chains. We remark that intramolecular association between vicinal hydroxyl groups contributes to the g(r) in the gOH-HO case. The peaks beyond r»3Å angstroms in both curves are due to the intramolecular association between the hydroxyl groups. A snapshot of the final conformation after 1000 ps MD trajectory is shown in Fig. 6.
Figure 6. Snapshot of the MD trajectory for the 2 chains system after a 1000 ps run. Different colour are used to distinguish the chains. Hydrogen atoms are omitted. Click inside the figure to retrieve the pdb file.
The chains are linked together forming a helix with a rough twofold screw symmetry where the stable junction zones consist of hydrogen bridges. A movie (0.4M) of the first 100 ps trajectory shows the process of the chain folding.
Click here to retrieve a 100 ps mpeg movie of the trajectory
A snapshot from the final step of a trajectory of the 3 chain system is shown in Fig. 7. The oxygen atoms in the same chains have the same colour.
Figure 7. Snapshot from the MD trajectory of the 3 chains system after a 1000 ps run. Different colour are used to distinguish the oxygen atoms of different chains. Hydrogen atoms are omitted. Click inside the figure to retrieve the pdb file.
The PGA chains are folded in the middle and exhibit a strong interchain interaction which leads to the folding of the chains onto themselves in a way which closely resembles the so called egg and box model [21]. The g(r) between the carboxylic oxygen atoms are shown in Fig. 8 and compared with those computed for the 1 and 2 chain systems. The overall conformation forms a three dimensional network. These findings agree with the models [22-23] proposed to explain the gel formation in pectic substances.
Figure 8. Radial Distribution Functions between the carboxyl (O2) oxygen atoms in the three different investigated systems.
The pattern of the g(r) emphasizes the importance of the carboxylate groups in the interchain interaction. The peaks, at r»5Å, are due to the carboxyl groups in the neighbouring monomer units, while the peaks at r»8-10Å originate from the carboxyl groups in the n and n+2 monomers. Both the two and three chain systems show a peak at r»3Å which indicates the formation of the hydrogen bonds between the carboxylate groups of different chains..
The results of the MD experiments show good qualitative agreement with the reports about gel formation by PGA chains in strong acidic media. The analysis of the g(r) provides evidence that the collapse of the PGA chains is mainly due to the formation of interchain hydrogen bonds.
Thanks are due to CINECA (grant n. 95/565-5) and to the MURST for the financial support.
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