Water is the dominant absorber of terahertz (THz) radiation in any hydrated biological object, which long made water absorption an obstacle to THz spectroscopy of biomolecules. The 'water as main target' program of THz biophotonics inverts this: because biomolecules restructure and retard the dynamics of surrounding water, the THz response of this hydration water should itself carry quantitative information about biomolecular structure and dynamics. What remains open is a validated, generally accepted methodology for separating and quantitatively modeling the biomolecular contribution, the dynamically perturbed hydration-water contribution, and the bulk-water background in measured THz spectra of aqueous biomolecular systems - including the non-additivity of protein-water cross-correlations - so that processing and interpretation of THz spectra of biomolecule solutions rest on generally accepted approaches rather than model-dependent ones.

Background

Terahertz spectroscopy measures the interaction of matter with electromagnetic radiation at frequencies of roughly 0.1-10 THz, where absorption is governed by picosecond-scale collective motion, and terahertz time-domain spectroscopy (THz-TDS) directly records both absorption and the complex dielectric permittivity, the frequency-dependent response of a medium to an applied electric field. In liquid water, the THz dielectric response is conventionally modeled as a superposition of Debye-type orientation-relaxation bands (reorientation of hydrogen-bonded and of more freely rotating molecules) plus a broad intermolecular stretch band near 200 cm^-1 arising from hydrogen-bond vibrations. Because water absorbs THz radiation extremely strongly, hydrated biomolecules were historically studied as dry powders or thin films; total-internal-reflection (attenuated total reflection) geometries made it possible to study proteins and nucleic acids in aqueous solution despite the water background.

A turning point was the realization that the water signal itself is informative: Heugen et al. (PNAS 2006) showed by THz absorption that dissolved sugars retard the dynamics of a 'dynamic hydration shell' extending well beyond the first water layer, and Ebbinghaus et al. (JACS 2008) showed the THz hydration response of peptides depends on amino-acid sequence and pH. Molecular dynamics decompositions (numerical simulations of atomic motion under an empirical force field) by Heyden and Tobias (PRL 2013) demonstrated that the protein-induced changes in the THz response of water are collective and extend spatially, so the measured spectrum is not a simple sum of independent solute and solvent parts. Reviews of THz spectroscopy of biological molecules (Cherkasova et al., 2009) and of the critical effect of hydration on THz signatures of biomolecules (Brown and Zhang, 2017) frame hydration-shell spectroscopy as the fundamental developing direction of the field, alongside applied diagnostics.

The central technical obstacle is that THz spectra of aqueous biomolecular solutions are essentially featureless: there are no pronounced bands, so all quantitative information must be extracted through a model. Three decompositions compete. First, an additive absorption model writes the solution absorption as a volume-weighted sum of biomolecule, hydration-shell, and undisturbed-water absorption; this is acknowledged to be rough, because the hydration shell is not a separate phase with a sharp boundary. Second, for dielectric permittivity spectra the additive approach fails in principle, and effective-medium models - Maxwell-Garnett, Bruggeman, and Landau-Lifshitz-Looyenga mixing formulas that treat the solution as inclusions in a continuous medium - must be used to separate the dielectric contributions of phases; a 2023 field review (Penkov, Biophysical Reviews) notes that despite its importance this separation is ignored in most work. Third, molecular dynamics simulations can decompose the response, and they show the decomposition is non-additive: protein and water dipole fluctuations are partially antiparallel and reduce the total absorption (Pandey and Cifra, 2026, for kinesin, a motor protein that moves along microtubules). In the related setting of hydrated crystals, THz vibrational modes of water-containing molecular crystals computed with harmonic density functional theory (a quantum-chemical electronic-structure method) require ad hoc frequency scaling, attributed to vibrational anharmonicity - the departure of the potential energy surface from a quadratic form, which couples modes and shifts frequencies - that is significant when water is in the lattice (Lee et al., 2020, cytosine and cytosine monohydrate).

Problem Statement

Can the terahertz response of water in and around biomolecules be made a quantitative probe of biomolecular structure and dynamics? Concretely: establish a validated, quantitative methodology for decomposing measured THz responses - absorption spectra and complex dielectric permittivity spectra (the frequency-dependent response of a medium to an applied electric field, resolved into real and imaginary parts) - of biomolecules in aqueous solution (including model membranes and cell suspensions measured in aqueous medium) into the biomolecular contribution, the dynamically perturbed hydration-water contribution (water whose rotational and hydrogen-bond dynamics are altered by the biomolecule), and the bulk-water background. A complete answer must (i) handle the non-additivity of biomolecule-water cross-correlations rather than assume an additive superposition of independent parts, (ii) supply effective-medium or equivalent dielectric mixing treatments (models that treat the heterogeneous solution as one phase embedded in another, effectively continuous phase) appropriate to permittivity spectra, and (iii) demonstrate on specified biomolecular systems that the extracted hydration-shell parameters reproduce independently known changes of biomolecular structure or dynamics (for example conformational state, denaturation, ligand binding, or hydration-dependent phase transformation). The problem is stated without restriction to a particular molecule, frequency band, or method.

References

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