ACID AND THERMAL DENATURATION OF BARNASE INVESTIGATED BY MOLECULAR DYNAMICS SIMULATIONS

Abstract The transition in barnase from the native state to a partially unfolded conformation has been studied by molecular dynamics simulations with explicit water molecules at 360 K and low Ph(450 ps), and at 600 K and neutral pH (three simulations of 120, 250 and 200 ps each). The use of several simulations provides evidence that the results are not sensitive to initial conditions. To mimic low pH conditions, the acidic sidechains in barnase were neutralized and the two histidine residues were doubly protonated. Runs at 300 K showed that the solvated structures at low pH (300 ps) and neutral pH (310 ps) are very similar. The main structural differences involved the acidic residues, histidine residues, and the β-turn connecting strands 4 and 5. When the temperature is raised to 360 K at low pH and to 600 K at neutral pH the barnase molecule begins to unfold. The molecule rapidly expands (Rgchanges from 13.9 A to 15.3 A in 450 ps at 360 K and from 13.7 A to between 15.1 and 15.5 A in 120 ps at 600 K). However, the expansion is not uniform. In all the simulations, the chain termini, loops and the N-terminal parts of the main α-helix (helix 1) show a continuous and progressive unfolding. An essential step in the denaturation process is that the major α-helix (helix 1) separates from the β-sheet; this is coupled to the exposure of the principal hydrophobic core, many of whose of non-polar sidechains become solvated by hydrogen-bonded water molecules. The barnase-water interaction energy improves during unfolding at the expense of the barnase self-energy. The deterioration of the intramolecular van der Waals energy suggests that the rupture of the tight packing during the initial unfolding phase contributes to the energy barrier to the denaturation process. The mutationally well-analyzed Asp8-Arg110-Asp12 double salt-bridge on the barnase surface is found to be marginally stable in the folded form in the simulations. A Poisson-Boltzmann calculation indicates that the salt-bridge is unstable; this is probably due to an overestimate of the solvation energy. A detailed analysis of the main hydrophobic core reveals that increase in solvent-accessible surface area and penetration of water molecules are simultaneous in the high-temperature simulation; at lower temperatures there is significant cavity formation and the entrance of the water molecules is somewhat delayed. The cavities occur in the neighborhood of the hydrophobic sidechains; the region formed by the sidechains of Val10, Leu14, Leu20, Tyr24, Ala74, Ile76 and Tyr90 is involved. The loosening of the core packing is coupled to an increase in the number of dihedral transitions. Water molecules connected in chains or clusters penetrate the cores by participating in hydrogen bonds with polar groups; e.g. on tyrosine and tryptophan sidechains. The hydrogen-bonding propensity of the water molecules tends to be satisfied throughout the denaturation process. This is evident also in the denaturation of the secondary structural elements, where water molecules compete with the interstrand and intrahelical hydrogen bonds. In all simulations, the β-sheet disruption starts at the edges and is coupled to an increase in the twist. In agreement with experimental results, α-helix 1 starts to denature at the N-terminal end. Helices 2 and 3 undergo early unfoldings at 600 K and neutral pH, while they are stable at 360 K and low pH. These differences arise from the greater entropic contribution to the unfolded state at high temperature. The overall similarity between the low-pH and high-temperature simulations indicates that the present results are representative of the barnase unfolding process. Experimental tests of the role of the solvent in barnase unfolding are proposed.