Functional State Dependence of Picosecond Protein Dynamics

We examine temperature dependent picosecond dynamics as a function of structure and function for lysozyme and cytochrome c using temperature dependent terahertz permittivity measurements. A double Arrhenius temperature dependence with activation energies E1 ~ 0.1 kJ/mol and E2 ~10 kJ/mol fits the native state response. The higher activation energy is consistent with the so-called protein dynamical transition associated with beta relaxations at the solvent-protein interface. The lower activation energy is consistent with correlated structural motions. When the structure is removed by denaturing the lower activation energy process is no longer present. Additionally the lower activation energy process is diminished with ligand binding, but not for changes in internal oxidation state. We suggest that the lower energy activation process is associated with collective structural motions that are no longer accessible with denaturing or binding.

💡 Research Summary

This study investigates how picosecond‑scale dynamics of proteins depend on their structural and functional states by employing temperature‑dependent terahertz (THz) dielectric spectroscopy. The authors focus on two well‑characterized model proteins: hen egg‑white lysozyme (HEWL) and cytochrome c (Cyt c). For each protein they examine the native form, a denatured form (induced by 6 M guanidinium chloride), a ligand‑bound form (HEWL complexed with tri‑N‑acetyl‑D‑glucosamine, 3NAG), and, for Cyt c, both oxidized (ferri) and reduced (ferro) states.

Measurements were performed on aqueous solutions in the 0.2–2.0 THz frequency range while varying temperature from roughly 100 K to 300 K. The primary observable is the imaginary part of the permittivity (ε″), which reflects the absorptive component of the dielectric response. Across all native samples, ε″ increases monotonically with temperature and exhibits a pronounced curvature change near 200 K, a signature commonly associated with the so‑called dynamical transition (DT) observed in many hydrated biomolecules.

The temperature dependence of ε″ can be quantitatively described by a sum of two Arrhenius terms:
ε″(T) = A₁ exp(−E₁/k_BT) + A₂ exp(−E₂/k_BT).
For native HEWL and Cyt c the extracted activation energies are E₁ ≈ 0.1 kJ mol⁻¹ and E₂ ≈ 10 kJ mol⁻¹. The higher‑energy component (E₂) remains essentially unchanged upon denaturation, ligand binding, or redox alteration, indicating that it originates from local β‑relaxations of water molecules at the protein surface—processes that give rise to the DT. The lower‑energy component (E₁) disappears or is strongly attenuated when the protein’s three‑dimensional structure is disrupted (denaturation) or when a ligand occupies the active cleft (HEWL‑3NAG). This behavior suggests that E₁ reflects collective, large‑scale motions of the protein backbone (“hinge” or “breathing” modes) that are only accessible in the native, structurally intact state.

Denatured samples display larger absolute values of ε″ than their native counterparts, which the authors attribute to an increase in “biological water” – water that is more strongly coupled to the protein surface and possesses a higher THz permittivity than bulk water. Ligand binding reduces ε″, consistent with a modest decrease in exposed surface area and possible suppression of the low‑energy collective motions.

Cytochrome c’s redox state (ferri vs. ferro) does not significantly affect either activation energy, reinforcing the interpretation that E₁ is tied to protein structure rather than to changes in the hydration shell induced by oxidation. The high‑energy E₂ values for all systems (~10–12 kJ mol⁻¹) align well with previous neutron quasielastic scattering and dielectric relaxation studies of the DT.

Overall, the work demonstrates that THz time‑domain spectroscopy, when combined with temperature‑dependent analysis, can separate contributions from solvent dynamics and intrinsic protein motions. The identification of a low‑energy, structure‑dependent activation process provides experimental support for the existence of thermally activated collective motions that may be functionally relevant at physiological temperatures. The authors propose that future investigations should extend this methodology to other proteins, protein‑protein complexes, and perhaps to monitor functional cycles in real time, thereby deepening our understanding of the dynamic underpinnings of biomolecular activity.