Backbone relaxation coupled to the ionization of internal groups in proteins: a self-guided Langevin dynamics study

Abstract

Pathways of structural relaxation triggered by ionization of internal groups in staphylococcal nuclease (SNase) were studied through multiple self-guided Langevin dynamics (SGLD) simulations. Circular dichroism, steady-state Trp fluorescence, and nuclear magnetic resonance spectroscopy have shown previously that variants of SNase with internal Glu, Asp, and Lys at positions 66 or 92, and Arg at position 66, exhibit local reorganization or global unfolding when the internal ionizable group is charged. Except for Arg-66, these internal ionizable groups have unusual pKa values and are neutral at physiological pH. The structural trends observed in the simulations are in general agreement with experimental observations. The I92D variant, which unfolds globally upon ionization of Asp-92, in simulations often exhibits extensive hydration of the protein core, and sometimes also significant perturbations of the beta-barrel. In the crystal structure of the V66R variant, the beta1 strand from the beta-barrel is domain-swapped; in the simulations, the beta1 strand is sometimes partially released. The V66K variant, which in solutions shows reorganization of six residues at the C-terminus of helix alpha1 and perturbations in the beta-barrel structure, exhibits fraying of three residues of helix alpha1 in one simulation, and perturbations and partial unfolding of three beta-strands in a few other simulations. In sharp contrast, very small structural changes were observed in simulations of the wild-type protein. The simulations indicate that charging of internal groups frequently triggers penetration of water into the protein interior. The pKa values of Asp-92 and Arg-66 calculated with continuum methods on SGLD-relaxed structures reached the normal values in most simulations. Detailed analysis of accuracy and performance of SGLD demonstrates that SGLD outperforms LD in sampling of alternative protein conformations without loss of the accuracy and level of detail characteristic of regular LD.

FIGURE 1

Crystal structure of SNase (60) and position of Lys-66 (red), Arg-66 (green), and Asp-92 (blue) side chains.

FIGURE 2

Percentage of total simulation time that residues in WT (black), V66K (red), V66R (green), or I92D (blue) spent in an α-helical or a β-strand conformation. Secondary structure was assigned with DSSP (top graph) and KAKSI (middle graph). Bottom graph shows average RMSD values for backbone atoms of each residue.

FIGURE 3

Snapshots from SGLD simulations representative of observed secondary structure changes. The residues undergoing change in the secondary structure are shown in red. The substituted ionizable groups are shown in stick representation.

FIGURE 4

(Top) Number of water molecules within a 6 Å radius of side-chain polar atoms of substituted ionizable groups, averaged over 1 ns. (Graphs from top to bottom) Lys-66, Arg-66, and Asp-92. Different colors represent different simulations. (Bottom) Snapshots from simulation of I92D variant when Asp-92 is neutral (left) and charged (right). Red spheres represent internal water molecules.

FIGURE 5

Energetic analysis of simulation D2 with the I92D variant. (From top to bottom) Total effective energy, self-energy, electrostatic interaction energy, number of solvent molecules around Asp-92, Born term, and pK_a value of Asp-92. All energies are in kcal/mol. All numbers represent averages over 1 ns of SGLD runs.