DNA heats up : Energetics of genome ejection from phage revealed by isothermal titration calorimetry

Most bacteriophages are known to inject their double-stranded DNA into bacteria upon receptor binding in an essentially spontaneous way. This downhill thermodynamic process from the intact virion toward the empty viral capsid plus released DNA is made possible by the energy stored during active packaging of the genome into the capsid. Only indirect measurements of this energy have been available until now using either single-molecule or osmotic suppression techniques. In this paper, we describe for the first time the use of isothermal titration calorimetry to directly measure the heat released (or equivalently the enthalpy) during DNA ejection from phage lambda, triggered in solution by a solubilized receptor. Quantitative analyses of the results lead to the identification of thermodynamic determinants associated with DNA ejection. The values obtained were found to be consistent with those previously predicted by analytical models and numerical simulations. Moreover, the results confirm the role of DNA hydration in the energetics of genome confinement in viral capsids.

💡 Research Summary

This paper presents the first direct measurement of the thermodynamics of DNA ejection from bacteriophage λ using isothermal titration calorimetry (ITC). By titrating phage particles into a solution containing the purified LamB receptor, the authors recorded the exothermic heat flow associated with genome release at constant temperature. Experiments were performed on two λ variants—wild‑type (48.5 kbp) and a shortened mutant (37.7 kbp)—across a temperature range of 22 °C to 32 °C. After correcting for mixing, dilution, and buffer‑buffer contributions, the net ejection enthalpy (ΔH_ej) was determined to be –(1.9 ± 0.3) × 10⁻¹⁶ J per virion for the wild type and –(5.3 ± 0.8) × 10⁻¹⁷ J per virion for the shorter genome. The enthalpy scales with genome length, confirming that ITC can sensitively detect differences in stored internal energy.

The measured values were compared with two theoretical frameworks. First, the inverse‑spool model, which treats the packaged DNA as an idealized spool and includes bending and short‑range DNA‑DNA repulsion, predicts ejection enthalpies of –1.9 × 10⁻¹⁶ J (48.5 kbp) and –6.1 × 10⁻¹⁷ J (37.7 kbp) under the same ionic conditions, matching the experimental data. Second, molecular dynamics simulations of DNA packaging (Petrov et al.) provide internal energies of +1.7 × 10⁻¹⁶ J (full genome) and +5.2 × 10⁻¹⁷ J (short genome). The near‑equal magnitude but opposite sign of these values relative to the measured ΔH_ej supports the hypothesis that the enthalpy of ejection mirrors the internal energy required for packaging, assuming negligible dissipative losses.

Temperature dependence of ΔH_ej revealed a modest decrease in magnitude with increasing temperature. Using the thermodynamic relations ΔG = ΔH – TΔS and ΔS = (ΔH – ΔG)/T, the authors estimated that the free energy change (ΔG) is roughly an order of magnitude smaller than the enthalpy, implying a positive entropy contribution (ΔS > 0). This counter‑intuitive result is explained by the release of ordered water molecules from the DNA hydration shells as the genome becomes densely packed; dehydration increases the overall disorder of the system, outweighing the loss of conformational entropy due to confinement. Similar hydration‑driven entropy gains have been reported in osmotic stress studies of dense hexagonal DNA phases.

Overall, the study demonstrates that ITC provides a quantitative, direct probe of the energetic landscape of viral genome ejection, complementing previous force‑based and osmotic suppression techniques. It confirms that short‑range DNA‑DNA interactions, particularly hydration forces, dominate the energetics of DNA confinement in phage capsids. The methodology can be extended to other viral systems, offering valuable insights for the design of antiviral strategies and synthetic nanocontainers.