Protein structure and dynamics in nonaqueous solvents: insights from molecular dynamics simulation studies

Abstract

Protein structure and dynamics in nonaqueous solvents are here investigated using molecular dynamics simulation studies, by considering two model proteins (ubiquitin and cutinase) in hexane, under varying hydration conditions. Ionization of the protein groups is treated assuming "pH memory," i.e., using the ionization states characteristic of aqueous solution. Neutralization of charged groups by counterions is done by considering a counterion for each charged group that cannot be made neutral by establishing a salt bridge with another charged group; this treatment is more physically reasonable for the nonaqueous situation, contrasting with the usual procedures. Our studies show that hydration has a profound effect on protein stability and flexibility in nonaqueous solvents. The structure becomes more nativelike with increasing values of hydration, up to a certain point, when further increases render it unstable and unfolding starts to occur. There is an optimal amount of water, approximately 10% (w/w), where the protein structure and flexibility are closer to the ones found in aqueous solution. This behavior can explain the experimentally known bell-shaped dependence of enzyme catalysis on hydration, and the molecular reasons for it are examined here. Water and counterions play a fundamental and dynamic role on protein stabilization, but they also seem to be important for protein unfolding at high percentages of bound water.

FIGURE 1

Active site of cutinase. Represented are the side chains of Asp 175, His 188, Ser 120, and Gln 121 (the last two contain the main chain between them). Arrows indicate the oxyanion hole, namely the NH groups of Gln 121 and Ser 42, and the OH group of Ser 42. The conformation A for Ser 120 is the one represented. Figure prepared with XtalView (McRee, 1992) and Raster3D (Merritt and Bacon, 1997).

FIGURE 2

Structural analysis in hexane simulations with different hydration. (a), r.m.s. deviation of Cα atoms of cutinase from average structure of the control in water. (b), Same as a for ubiquitin, but the C-terminal tail (residues 71–76) is excluded. (c), r.m.s. deviation (of the 2–4 ns average) of the active site residues of cutinase, when compared with the x-ray structure. The atoms contributing for the r.m.s. deviation are specified in Footnote 3 of Table 1. The dashed line represents the average r.m.s. deviation observed in the water simulation. (d) Percentage of cutinase residues (2–4 ns average) in the same secondary structure class as in the control water simulation without counterions (using the 2–4 ns average). See Footnote 5 of Table 1 for the secondary structure types considered. (e), The same as d, but for ubiquitin. (f), Hydrogen bond analysis for cutinase (using the 2–4 ns period). See Footnote 6 of Table 1. Persistent hydrogen bonds are the ones occurring more than 50% of the time. The dashed line represents the number of hydrogen bonds observed in the corresponding water simulation. (g), Same as f, but for ubiquitin.

FIGURE 3

r.m.s. fluctuations analysis of hexane simulations (corresponding to the 2–4 ns period). (a), r.m.s. fluctuations for the cutinase protein atoms (all atoms). The dashed line represents the average r.m.s. fluctuation observed in the corresponding water simulation. (b), Same as a, but for ubiquitin (the C-terminal tail—residues 71–76—is not included). (c), r.m.s. fluctuations of the counterions in the cutinase simulations. (d), Same as c, but for ubiquitin. (e), r.m.s. fluctuations for water molecules in cutinase simulations. (f), Same as e, but for ubiquitin.

FIGURE 4

Average number of water clusters (corresponding to the 2–4 ns span). These clusters are constituted by loosely interacting water molecules. The criterion used was to group water molecules whose oxygen atoms are within 5 Å of any other water oxygen of the cluster. Clusters of only one element are not considered. The results for cutinase are represented by filled squares (upper part) whereas ubiquitin results are represented by filled triangles (lower part).

FIGURE 5

Stereo pictures of the final conformation of cutinase in several simulation conditions after 4 ns (the simulations corresponding to the median are selected here). The fold is represented by a smooth blue trace and the side chains of the active site are represented using sticks. Sodium and chloride ions are rendered using spheres, with red and yellow colors respectively. In the simulations in hexane containing water, water molecules are rendered using sticks. The figures were prepared using Molscript (Kraulis, 1991) and Raster 3D (Merritt and Bacon, 1997). Presented are simulations in pure water (a), and in hexane with ions and 0% (b), 5% (c), 10% (d), and 25% (e) of water.