Water-peptide site-specific interactions: a structural study on the hydration of glutathione

Abstract

Water-peptide interactions play an important role in determining peptide structure and function. Nevertheless, a microscopic description of these interactions is still incomplete. In this study we have investigated at the atomic scale length the interaction between water and the tripeptide glutathione. The rationale behind this work, based on the combination between a neutron diffraction experiment and a computer simulation, is twofold. It extends previous studies on amino acids, addressing issues such as the perturbation of the water network brought by a larger biomolecule in solution. In addition, and more importantly, it seeks a possible link between the atomic length scale description of the glutathione-water interaction with the specific biological functionality of glutathione, an important intracellular antioxidant. Results indicate a rather weak hydrogen bond between the thiol (-SH) group of cysteine and its first neighbor water molecule. This -SH group serves as a proton donor, is responsible for the biological activity of glutathione, and it is involved in the formation of glutathione disulfide, the oxidized form of glutathione. Moreover, the hydration shell of the chemically identical carboxylate group on the glutamic acid residue and on the glycine residue shows an intriguing different spatial location of water molecules and coordination numbers around the two CO2(-) groups.

Figure 1

Molecular structure of the tripeptide glutathione (GSH) in its protonation state ${\alpha }_1$ (top), and ${\alpha }_{12}$ (bottom). GSH-${\alpha }_1$ and GSH-${\alpha }_{12}$ represent, respectively, the 60% and 30% of the possible states of GSH at the investigated pH $\sim 3$. Each atom site has been labeled according to the symbols used in the EPSR simulation, see Table S2 in the Supporting Material. To see this figure in color, go online.

Figure 2

Site-site radial distribution function of water sites, O_w and H_w, for water as a solvent (thick solid lines) compared with pure water (thin solid lines). The insert shows the region around the second peak of the O_w-O_w. The peak position is unchanged, compared with pure water, indicating that the solute GSH does not alter average water structure.

Figure 3

Site-site radial distribution function of water oxygen O_w around the oxygens of the glutamic acid, O_g, and the oxygens on the glycine, O₁ and O₂ for GSH-${\alpha }_1$, see Fig. 1. The O_w-O_w radial distribution function for pure water is also shown as a thin solid line for comparison. The insert shows the region of the first peak of all the plotted radial distribution functions (RDFs), indicating a clear shift to smaller distances of its position for the O_g-O_w, O₁-O_w, and O₂-O_w RDFs, in comparison with pure water. All glutathione-water RDFs show a slope, that is the signature of excluded volume effects, because of the large size of the tripeptide compared with that of a water molecule.

Figure 4

Site-site radial distribution function of water hydrogen, H_w, around the oxygens of the glutamic acid, O_g (thick solid line). For the sake of comparison, the O_w-H_w is also plotted as a thin solid line. The first peak, usually taken as the signature of the H-bond, is slightly shifted to smaller distances for O_g-H_w, suggesting the presence of shorter and possibly stronger H-bonds between water and O_g sites of glutathione. Excluded volume effects, because of the large size of the tripeptide, compared with that of a water molecule, are visible for the plotted O_w-H_w RDF.

Figure 5

Spatial density functions (SDF) showing the distribution of water molecules around the -${\text{CO}}_2^{-}$ group on glutamic acid (A), and around the -${\text{CO}}_2^{-}$ group on glycine (B). The yellow shaded areas represents regions where there is a probability of finding a water molecule at a distance range 2.00 to 4.47 Å (A) or at a distance range 2.00 to 4.26 Å (B) from the central carbon atom. These distance ranges correspond to the first coordination shell of the C_cg-O_w RDF, and of the C_c-O_w RDF, respectively (data not shown), where C_cg is the carbon atom of the -${\text{CO}}_2^{-}$ group on glutamic acid, and C_c is the carbon atom on glycine (see Fig. 1). The plotted SDFs show 65% of the water molecules within the ranges indicated. To see this figure in color, go online.

Figure 6

Top panel: site-site radial distribution function of water oxygen O_w around the oxygens, O_1h and O_2h, of the carboxylic group of glycine in the GSH-${\alpha }_{12}$ protonation state, see Fig. 1. The O_w-O_w radial distribution function for pure water is also shown as a thin solid line for comparison. The insert shows the region of the first peak of all the plotted RDFs, indicating a slight shift to a larger distance of its position for the O_1h-O_w, whereas there is no change and O₁-O_w, and no variation of its position for O_2h-O_w RDFs, in comparison with pure water. Bottom panel: spatial density functions (SDF) showing the distribution of water molecules around the -COOH group on glycine in the GSH-${\alpha }_{12}$ protonation state, see Fig. 1. The yellow shaded areas represents regions where there is a probability of finding a water molecule at a distance range 2 to 4.44 Å from the central carbon atom. This distance range corresponds to the first coordination shell of the C_cch-O_w RDF (data not shown), where C_cch is the carbon atom of the -COOH group on glycine (see Fig. 1). The plotted SDFs show 65% of the water molecules within the ranges indicated. To see this figure in color, go online.

Figure 7

Top panel: site-site radial distribution function of water oxygen, O_w, around the hydrogens of the amine group on the glutamic acid, H_x (thick solid line). For the sake of comparison, the H_w-O_w is also plotted as a thin solid line. The first peak, usually taken as the signature of the H-bond, is clearly shifted to smaller distances for H_x-O_w, suggesting the presence of shorter and possibly stronger H-bonds between water and H_x sites of glutathione. Notice the slope, because of excluded volume effects, of the plotted RDF. Bottom panel: spatial density functions (SDF) showing the distribution of water molecules around the -NH³⁺ group on glutamic acid, see Fig. 1. The yellow shaded areas represents regions where there is a probability of finding a water molecule at a distance range 1 to 3.57 Å from the central nitrogen atom. This distance range corresponds to the first coordination shell of the N-O_w RDF (data not shown). The plotted SDFs show 70% of the water molecules within the ranges indicated. To see this figure in color, go online.

Figure 8

Site-site radial distribution function of water oxygen, O_w, around the hydrogen of the -SH group on cysteine, H_s (thick solid line). For the sake of comparison, the H_w-O_w is also plotted (thin solid line). The first peak, usually taken as the signature of the H-bond, is clearly reduced in amplitude and slightly shifted to larger distances for H_x-O_w, indicating a relatively low affinity for water of this highly reactive group of glutathione. The sloping behavior of the H_x-O_w RDF can be attributed to the presence of excluded volume effects.