The volume changes of unfolding of dsDNA

Abstract

High hydrostatic pressure can have profound effects on the stability of biomacromolecules. The magnitude and direction (stabilizing or destabilizing) of this effect is defined by the volume changes in the system, ΔV. Positive volume changes will stabilize the starting native state, whereas negative volume changes will lead to the stabilization of the final unfolded state. For the DNA double helix, experimental data suggested that when the thermostability of dsDNA is below 50°C, increase in hydrostatic pressure will lead to destabilization; i.e., helix-to-coil transition has negative ΔV. In contrast, the dsDNA sequences with the thermostability above 50°C showed positive ΔV values and were stabilized by hydrostatic pressure. In order to get insight into this switch in the response of dsDNA to hydrostatic pressure as a function of temperature, first we further validated this trend using experimental measurements of ΔV for 10 different dsDNA sequences using pressure perturbation calorimetry. We also developed a computational protocol to calculate the expected volume changes of dsDNA unfolding, which was benchmarked against the experimental set of 50 ΔV values that included, in addition to our data, the values from the literature. Computation predicts well the experimental values of ΔV. Such agreement between computation and experiment lends credibility to the computation protocol and provides molecular level rational for the observed temperature dependence of ΔV that can be traced to the hydration. Difference in the ΔV value for A/T versus G/C basepairs is also discussed.

Graphical abstract

Figure 1

Hypothetical thermodynamic cycle for computing the volume changes of unfolding of dsDNA in aqueous solution, ΔV_Tot. Considering that the volume is a state function, the native dsDNA can be transferred into the gas phase. This process will be accompanied by changes in the volume of hydration of the native state V_Hyd,N. The dsDNA, in the gas phase, is then unfolded into two single-stranded DNA molecules. On this step, the relevant volume change for the overall thermodynamic cycle will be represented by ΔV_void. Finally, the two unfolded single-stranded DNA molecules will be transferred back from gas phase into the aqueous solution. This process will be accompanied by the change in the interactions between solvent water and DNA and corresponds to the volume of hydration of unfolded state, V_Hyd,U1 and V_Hyd,U2. Thus the net change in volume in aqueous solution will be ΔV_Tot= ΔV_void + (V_Hyd,U1 + V_Hyd,U2 – V_Hyd,N). To see this figure in color, go online.

Figure 2

Comparison of experimental and fitted (using Eq. 1) values of hydration volume for model compounds at 25°C. The correlation coefficient R² = 0.994 and the slope is 0.99 ± 0.01. Coefficients of the fit are listed in Table S3, and comparison of the experimental and fitted values at other temperatures are given in Table S2. To see this figure in color, go online.

Figure 3

Comparison of experimental and computed values for volume change upon unfolding of dsDNA. (A) Volume change upon double-stranded DNA unfolding, ΔV_exp, for over 50 different experimental data points compared with the value computed at the same temperature as the experiment. Red symbols are values computed from direct simulations (amber99bsc1 force field), and green symbols are for the polynucleotides, with values computed using simple additivity contributions derived for amber99bsc1 force field. Analogous plots for the nearest-neighbor (NN) and/or charmm27 force field show similar trends (see Figs. S7, S8, and S9). Error bars for each point are smaller than the symbols (please see Table S9 for actual estimated errors). (B) Dependence of volume change upon dsDNA unfolding, ΔV_Tot, on temperature. Blue circles, experimental data; red triangles, values computed from direct simulations using amber99bsc1 force field; green triangles, values computed using simple additivity contributions derived for amber99bsc1 force field (see Tables S5 and S7). The solid lines are calculated temperature dependences of volume changes using linear extrapolation of V_Hyd on temperature for AT (red) and GC (green) basepairs. The dashed lines show expected temperature dependencies if V_Hyd levels off at high temperatures. To see this figure in color, go online.

Figure 4

Contribution from changes in void volume, and hydration to the total volume changes of unfolding of AT or GC basepairs, at 20°C and 60°C. Gray bars, ΔV_void; blue bars, ΔV_hyd; dark-red bars, ΔV_Tot for AT basepair; dark-green bars, ΔV_Tot for GC basepair. To see this figure in color, go online.