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

The paper presents the first direct measurement of the thermodynamic heat released during bacteriophage λ genome ejection by employing isothermal titration calorimetry (ITC). Traditionally, the energy stored in viral capsids during DNA packaging has been inferred indirectly through single‑molecule force spectroscopy or osmotic suppression assays, leaving a gap in quantitative experimental data on the actual enthalpic change that accompanies DNA release. In this study, purified λ phage particles were mixed with a solubilized form of the LamB receptor protein in a controlled ITC cell. Upon receptor binding, the capsid undergoes a conformational transition that triggers the rapid ejection of the 48.5 kbp double‑stranded DNA into solution. The calorimeter records the minute heat flow associated with each injection, allowing the authors to integrate the signal and obtain the total enthalpy change (ΔH) per virion.

The measured average enthalpy of ejection is approximately –5.8 × 10⁻¹⁶ J per virion (≈ –14 kcal mol⁻¹), a value that aligns closely with predictions from analytical models and molecular dynamics simulations that estimate the stored packaging energy to be on the order of –15 kcal mol⁻¹. Temperature‑dependence experiments reveal that ΔH becomes less negative at higher temperatures, while the associated entropy change (ΔS) remains negative, indicating that the ejection is enthalpy‑driven but accompanied by an entropy loss, primarily due to the re‑ordering of water molecules. By varying ionic strength (0.1–1 M NaCl) and Mg²⁺ concentration (0–10 mM), the authors demonstrate that higher ion concentrations diminish the magnitude of the heat released by roughly 20 %, supporting the notion that electrostatic screening and ion‑water interactions modulate the hydration shell of DNA and thus its contribution to the overall energetics.

A mechanistic model linking the measured ΔH to the internal capsid pressure is also presented. The pressure, generated by DNA bending stress, electrostatic repulsion, and confinement entropy, drops sharply as the genome exits, converting stored mechanical energy into thermal energy. The model, which incorporates capsid elasticity and DNA persistence length, reproduces the experimental enthalpy values within a 5 % error margin, confirming that the dominant source of heat is the relaxation of DNA‑induced stress rather than purely chemical binding events.

The authors discuss methodological advantages of ITC: it provides a bulk, non‑destructive readout of the entire ejection process in a single experiment, avoiding the need for labeling or external force application. Limitations include the requirement to fine‑tune phage and receptor concentrations to keep the heat signal within the instrument’s dynamic range, as excessively rapid ejection can lead to signal saturation. Nonetheless, the approach is readily adaptable to other dsDNA phages (e.g., T4, P22) and to synthetic nanocontainers designed for drug delivery, where understanding the balance between stored mechanical energy and hydration effects is crucial for controlled release.

In summary, this work validates long‑standing theoretical predictions about the energetics of viral genome confinement, highlights the pivotal role of DNA hydration in governing the enthalpic component of ejection, and establishes ITC as a powerful tool for probing the thermodynamics of viral infection and nanocapsid engineering.