Preferential interactions of guanidinum ions with aromatic groups over aliphatic groups

Abstract

Small angle neutron scattering (SANS) and molecular dynamics (MD) simulations were used to characterize the long-range structuring (aggregation) of aqueous solutions of isopropanol (IPA) and pyridine and the effect on structuring of guanidinium chloride (GdmCl). These solutes serve as highly soluble analogs of the nonpolar aliphatic (IPA) and aromatic (pyridine) side chains of proteins. SANS data showed that isopropanol and pyridine both form clusters in water resulting from interaction between nonpolar groups of the solutes, with pyridine aggregation producing longer-range structuring than isopropanol in 3 m solutions. Addition of GdmCl at 3 m concentration considerably reduced pyridine aggregation but had no effect on isopropanol aggregation. MD simulations of these solutions support the conclusion that long-range structuring involves hydrophobic solute interactions and that Gdm(+) interacts with the planar pyridine group to suppress pyridine-pyridine interactions in solution. Hydrophobic interactions involving the aliphatic groups of isopropanol were unaffected by GdmCl, indicating that the planar and weakly hydrated Gdm(+) cation cannot make productive interactions with the highly curved or "lumpy" aliphatic groups of this solute. These observations support the conclusion that the effects of Gdm(+) ions on protein-stabilizing interactions involving aromatic amino acid side chains make significant contributions to the denaturant activity of GdmCl, whereas interactions with the "lumpy" aliphatic side chains are likely to be less important.

Figure 1

SANS data from: (a) 3 m pyridine in D₂O (open triangles), 3 m pyridine plus 3 m GdmCl in D₂O (open circles); and (b) 3 m isopropanol in D₂O (open triangles), 3 m isopropanol plus 3 m GdmCl in D₂O (open circles). Solid lines are model fits using the O-Z model. The addition of 3m GdmCl to pyridine clearly reduces the length scale of the aggregates, while GdmCl has no effect on the aggregates of isopropanol. The structureless scattering from D₂O (solid triangles), and 3 m GdmCl in D₂O (solid squares) are shown in each graph for comparison. Both graphs are on the same scale, and are in absolute units. The height of the symbols is representative of one standard deviation of the measured intensity.

Figure 2

The size and shape of the species used in this study. Shown on the left is isopropanol; on the middle left is the flat pyridine molecule. On the middle right is the flat denaturant guanidinium ion, and on the right is water, providing a visual reference of the comparative sizes. The top, middle and bottom rows show different orientations of these molecules. The molecular volumes of these molecules are 71, 78, 57 and 19 ų for isopropanol, pyridine, Gdm⁺ and water, respectively.

Figure 3

The probability of finding solute-solute coordination numbers for 3m isopropanol (left, blue), 3m isopropanol in 3m GdmCl (left, red), 3m pyridine (right, blue) and 3m pyridine in 3m GdmCl (red), as calculated from MD simulations. Shown in black in each panel is the solute-solute CN for Gdm⁺ in a 3 molal solution of GdmCl, a species known to show mild homo-ion association in a specific stacking type orientation that does not result in significant longer range structures.

Figure 4

Left: The probability of a solute being found in a cluster of size N in isopropanol solution (grey), and in an isopropanol/GdmCl solution (black). Right: The probability of a solute being found in a cluster of size N in pyridine solution (grey) and in a pyridine/GdmCl solution (black).

Figure 5

The upper row shows density maps of H_PY (white) and C_PY (red) for pyridine around pyridine in the GdmCl-free simulation. From left to right the contours denote number densities of 0.0250, 0.0175 and 0.0135 atoms Å⁻³. These density maps indicate that T-type pyridine-pyridine interactions are preferred (see Figure 7). The lower row shows density maps [H_Gdm (white), N_Gdm (purple)], for Gdm⁺ around pyridine. From left to right the N_Gdm have contours are 0.0150, 0.0105, 0.0081 atoms Å⁻³ while the H_Gdm contours have contours of double these values. This indicates that the Gdm⁺ ions tend to stack face-on to the pyridine, except when they hydrogen bond to the nitrogen of the pyridine, yielding a ‘headphones’ shaped density map.

Figure 6

A snap shot from the pyridine-GdmCl simulation, showing typical solute-solute interactions. The two left most pyridines (red) are showing a T-type interaction while the two Gdm⁺ ions (yellow) and one pyridine on the right are making stacking interactions.

Figure 7

Upper, the density map for C_IPA around isopropanol in the simulation without GdmCl (red), and with GdmCl (yellow). From left to right the density contours have values of 0.015, 0.012. 0.0088 atoms Å⁻³. The lower row shows the density map for N_Gdm (purple) and H_Gdm (white) around isopropanol (contours at 0.015, 0.012 and 0.0088 atoms Å⁻³, left to right, for N_Gdm, and at twice these values for H_Gdm). The dominant interaction seen between Gdm⁺ and isopropanol is the hydrogen bond with the hydroxyl oxygen of the isopropanol.