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Chirality-selective vibrational sum frequency generation (chiral SFG) spectroscopy has emerged as a powerful technique for the study of biomolecular hydration water due to its sensitivity to the induced chirality of the first hydration shell. Thus far, water O–H vibrational bands in phase-resolved heterodyne chiral SFG spectra have been fit using one Lorentzian function per vibrational band, and the resulting fit has been used to infer the underlying frequency distribution. Here, we show that this approach may not correctly reveal the structure and dynamics of hydration water. Our analysis illustrates that the chiral SFG responses of symmetric and asymmetric O–H stretch modes of water have opposite phase and equal magnitude and are separated in energy by intramolecular vibrational coupling and a heterogeneous environment. The sum of the symmetric and asymmetric responses implies that an O–H stretch in a heterodyne chiral SFG spectrum should appear as two peaks with opposite phase and equal amplitude. Using pairs of Lorentzian functions to fit water O–H stretch vibrational bands, we improve spectral fitting of previously acquired experimental spectra of model β-sheet proteins and reduce the number of free parameters. The fitting allows us to estimate the vibrational frequency distribution and thus reveals the molecular interactions of water in hydration shells of biomolecules directly from chiral SFG spectra.

Biomolecular hydration is fundamental to biological functions. Using phase-resolved chiral sum-frequency generation spectroscopy (SFG), we probe molecular architectures and interactions of water molecules around a self-assembling antiparallel β-sheet protein. We find that the phase of the chiroptical response from the O-H stretching vibrational modes of water flips with the absolute chirality of the (l-) or (d-) antiparallel β-sheet. Therefore, we can conclude that the (d-) antiparallel β-sheet organizes water solvent into a chiral supermolecular structure with opposite handedness relative to that of the (l-) antiparallel β-sheet. We use molecular dynamics to characterize the chiral water superstructure at atomic resolution. The results show that the macroscopic chirality of antiparallel β-sheets breaks the symmetry of assemblies of surrounding water molecules. We also calculate the chiral SFG response of water surrounding (l-) and (d-) LK₇β to confirm the presence of chiral water structures. Our results offer a different perspective as well as introduce experimental and computational methodologies for elucidating hydration of biomacromolecules. The findings imply potentially important but largely unexplored roles of water solvent in chiral selectivity of biomolecular interactions and the molecular origins of homochirality in the biological world.