Nonthermal acceleration of protein hydration by sub-terahertz irradiation

The study addresses how sub-terahertz (sub-THz) electromagnetic fields affect collective protein–water dynamics and whether these effects are thermal or nonthermal. Protein function depends critically on hydration layers, and water dynamics near biomolecular surfaces involve multiple relaxational and vibrational modes in the sub-THz region. Prior spectroscopies (optical Kerr-effect, extraordinary acoustic Raman, THz) and simulations have identified coupled motions of hydration water and protein surfaces at sub-THz frequencies. Reports using 0.1–0.5 THz sources indicate biological effects (changes in α-helix electron density, actin polymerization, gene expression, membrane permeability). Previous NMR work suggested 0.1 THz can induce protein dynamic changes opposite to heating, implying nonthermal components. However, mechanisms and energy retention in specific protein–water interactions are unclear. The authors propose using dielectric relaxation (DR) measurements of aqueous protein solutions to evaluate sub-THz-induced perturbations to rotational freedom of water dipoles and collective relaxations shaped by hydrogen-bond networks.

- Hydration water dynamics near proteins exhibit multiple components at sub-THz frequencies, involving relaxational and vibrational modes assigned to coupled water–protein surface motions. - Techniques: optical Kerr-effect, extraordinary acoustic Raman, and THz spectroscopies have advanced probing biomacromolecular dynamics in the THz region. - Biological effects of sub-THz irradiation (0.1–0.5 THz): changes in α-helix electron density in lysozyme crystals, effects on actin polymerization, gene expression, and membrane permeability. - Prior NMR study showed 0.1 THz induces structural dynamics in ubiquitin opposite to those from heating, suggesting nonthermal influences. - DR spectra of protein solutions contain β (~10 MHz), δ1 (~0.1 GHz), δ2 (~4 GHz) hydration relaxations, and bulk water γ1 (~20 GHz) and γ2 (150–600 GHz) components; temperature dependence of bulk water relaxations has been characterized in previous DR and THz-TDS studies, though small amplitudes and model dependencies complicate analysis.

- Experimental design: Developed a time-lapse microwave dielectric relaxation (DR) reflection method coupled with sub-THz irradiation. Used an open-ended coaxial probe and exploited standing wave effects in a short path-length sample cell (l = 1.0 mm) to sensitively detect changes in complex dielectric permittivity. - Sub-THz irradiation: 0.1 THz pulsed irradiation from a klystron, average power density 16 mW/cm², estimated electric field ~0.15 kV/m at the sample surface; pulse width ~0.8–1 µs. Irradiation applied from the side opposite to the VNA field to avoid interference (none detected). - Samples: Aqueous chicken egg white lysozyme solutions prepared by dissolving 30 mg or 100 mg powder in 1 mL pure water (2.9 or 9.1 wt%), typically measured ~2 h after dissolution; pH ~3.4. Pure water controls were also measured. Additional solutes for generality tests: cytochrome C, salmon sperm DNA, NaCl. - Temperature protocols: Sub-THz irradiation for 10 min raised sample temperature by ~4 °C due to water absorption. Control protocols: high-temperature control (HTC, ~+6 °C via Peltier), low-temperature control (LTC, ~−4 °C), and general control (GC) with the same setup but no THz power. After 10 min perturbation, dielectric responses were monitored for an additional 30 min as temperature relaxed back to 24 °C (~20 min recovery). - DR measurements: Vector network analyzers (100 MHz–14 GHz; extended to 43.5 GHz when needed) with open/short/standard calibrations. The same sample before (Standard) and after (Unknown) irradiation enabled precise time-lapse comparison. Complex permittivity extracted via reflection method; analysis focused on the bulk-water slow relaxation (γ1) component sufficiently separated in frequency. Nyquist plots were used to approximate a single Debye relaxation to extract εγ1(s), εγ1(∞), and relaxation frequency fγ1. Deviations from Debye behavior were quantified as Δr, and a sharp resonance-like peak PΔr around 7–8 GHz arising from standing wave fine-tuning (due to l ≈ λ/4) was used as a sensitive indicator of small permittivity changes. - THz time-domain spectroscopy (THz-TDS): ATR configuration (0.3–2.5 THz); imaginary part ε″ used preferentially due to lower error. For 28.6 wt% lysozyme, vibrational contributions assigned to lysozyme were subtracted (per literature), spectra normalized by water fraction, and compared to pure water across temperatures. Post-irradiation vs non-irradiated control spectra were differenced to assess changes overlapping γ2 relaxation. - NMR spectroscopy: 1H-13C HSQC at 800 MHz, 298 K, on 9.1 wt% lysozyme in 10% D2O. Timepoints: ~3–4 h and 24–25 h after dissolution following THz, HTC, or GC histories (THz/HTC applied 20–30 min after dissolution). Assessed chemical shift changes and methyl (CH3) signal intensity variations; pairwise correlation analyses of methyl intensities across conditions to infer conformational/hydration pathway differences; mapped significantly changing residues onto lysozyme crystal structure, with attention to hydrophobic cavity regions. - Data analysis: Debye model approximations via Nyquist-plane semicircle; extrapolation of εγ1(0) and εγ1(∞); correlation of PΔr with low-frequency (0.3–3 GHz) ε″ increases; comparison to THz-TDS ε″ changes in γ2 band to infer redistribution between fast (γ2) and slow (δ) water relaxation components.

- Sub-THz irradiation decreased the high-frequency limit of slow water relaxation ε1(∞) in lysozyme solutions more than expected from the modest temperature rise (~4 °C), indicating a nonthermal effect; this decrease did not occur in pure water and was dependent on lysozyme concentration (smaller at 2.9 wt% than at 9.1 wt%) and was more evident when extending measurement frequency beyond 14 GHz. - The presence of lysozyme reduced the temperature-driven difference in ε1(∞) (HTC vs LTC) observed in pure water to ~30%, and irradiation specifically perturbed fast water dynamics associated with protein–water interactions. - THz-TDS showed that, relative to pure water, lysozyme-interacting water exhibited increased ε″ in the 0.3–1 THz range overlapping γ2 relaxation, with temperature dependence opposite to that of pure water, suggesting distinct relaxation origin in the presence of lysozyme. After 0.1 THz irradiation, ε″ in the THz region, including the γ2 peak, slightly decreased compared to non-irradiated control, consistent with a reduction in fast (γ2) relaxation strength. - Time-lapse DR deviation signal PΔr (around 7–8 GHz) increased and shifted to higher frequency (~+1 GHz) following irradiation, even as temperature returned to baseline, indicating a gradual, irradiation-accelerated decrease in dielectric permittivity and an increase in low-frequency ε″ (0.3–3 GHz). This supports a shift from fast (γ2) to slow (δ) hydration water relaxation, interpreted as progression toward more hydrophobic hydration (more extensive H-bonding) around lysozyme. - NMR detected no significant chemical shift changes among THz, HTC, and GC, indicating no large structural rearrangements. Methyl intensity correlation analysis revealed that the THz state at ~3 h positively correlates with the GC state at 24 h (R ≈ 0.85), suggesting irradiation accelerates the pathway toward the 24 h hydrated state, whereas heating follows an opposite pathway relative to THz at 3 h (negative correlation). Residues with altered methyl intensities localized near the hydrophobic cavity, with deeper cavity changes more pronounced after THz at 3 h compared to GC at 24 h. - Generality tests: A similar PΔr response was observed for cytochrome C (amphiphilic protein with hydrophobic core), weaker for salmon sperm DNA (highly hydrophilic surface), and absent for NaCl, implying specificity to hydration near hydrophobic macromolecular surfaces. - Collectively, results indicate that 0.1 THz irradiation selectively excites fast hydration dynamics at protein surfaces and nonthermally accelerates the formation of hydrophobic hydration, reducing orientational polarization and dielectric permittivity over minutes to hours.

The findings demonstrate that sub-THz irradiation nonthermally perturbs hydration dynamics in protein solutions by selectively exciting fast water motions associated with protein–water interactions. In lysozyme solutions, irradiation caused a larger decrease in ε1(∞) than attributable to heating and induced a redistribution of relaxation strength from fast (γ2) to slow (δ) components, reflecting a shift toward more extensive hydrogen-bond networks characteristic of hydrophobic hydration. The time evolution of the sensitive PΔr signal shows that irradiation accelerates the slow, post-dissolution hydration processes leading to reduced dielectric permittivity and increased low-frequency ε″, consistent with the formation of more ordered hydration layers. NMR corroborates a nonthermal pathway: THz-exposed samples at ~3 h resemble the GC state at 24 h (R ≈ 0.85) without major structural changes, and the affected residues cluster around the hydrophobic cavity, indicating that the primary changes are in protein–water interactions rather than protein fold. The absence of similar effects in pure water and the dependence on hydrophobic macromolecular surfaces (cytochrome C vs DNA/NaCl) further support specificity to hydration at hydrophobic interfaces. Mechanistically, the excited fast hydration dynamics may enable water molecules to overcome strong ion-dipole interactions at the charged protein surface and reorganize H-bond networks, including entry into hydrophobic cavities, thereby shortening the timescale to reach hydrophobic hydration. Potential mechanisms include transient modification of energy barriers during the ~0.8 µs pulses or long-lived low-frequency collective modes (e.g., Fröhlich-type excitations), though the precise origin remains unresolved.

This work introduces a highly sensitive, time-lapse DR approach that leverages standing-wave enhancement in a short-path cell to monitor subtle, time-dependent changes in hydration dynamics under sub-THz irradiation. Combining DR with THz-TDS and NMR, the study provides evidence that 0.1 THz irradiation nonthermally accelerates the transition of lysozyme hydration toward a more hydrophobic, hydrogen-bonded state, decreasing dielectric permittivity and shifting relaxation strength from fast (γ2) to slow (δ) components. The effects are specific to protein systems with hydrophobic surfaces and are not reproduced in pure water or simple ionic solutions. These results highlight how modulation of collective hydration dynamics can influence protein environments and potentially functions. Future research should directly image and quantify the evolution of water H-bond networks over protein surfaces (including hydrophobic cavities) before and after irradiation, implement in situ NMR or other structural probes during irradiation, and elucidate the mechanisms and lifetimes of the excited modes responsible for the observed nonthermal effects.

- The mechanism by which sub-THz excitation energy remains localized in fast hydration dynamics and the duration of such localization are not clarified. - Direct observation of H-bond network reorganization at the protein surface was not performed; interpretations are inferred from DR and THz-TDS spectral changes. - Changes in ε′ carry larger measurement errors in THz-TDS; small effects approach measurement uncertainty. - While NMR shows no large-scale structural rearrangements, subtle conformational heterogeneity and counterion effects cannot be fully excluded. - Generalizability across diverse proteins and solution conditions (pH, ionic strength) requires further study.