Detecting Interplay of Chirality, Water, and Interfaces for Elucidating Biological Functions.

Chemists have long been fascinated by chirality, water, and interfaces, making tremendous progress in each research area. However, the chemistry emerging from the interplay of chirality, water, and interfaces has been difficult to study due to technical challenges, creating a barrier to elucidating biological functions at interfaces. Most biopolymers (proteins, DNA, and RNA) fold into macroscopic chiral structures to perform biological functions. Their folding requires water, but water behaves differently at interfaces where the bulk water hydrogen-bonding network terminates. A question arises as to how water molecules rearrange to minimize free energy at interfaces while stabilizing the macroscopic folding of biopolymers to support biological function. This question is central to solving many research challenges, including the molecular origin of biological homochirality, folding and insertion of proteins into cell membranes, and the design of heterogeneous biocatalysts. Researchers can resolve these challenges if they have the theoretical tools to accurately predict molecular behaviors of water and biopolymers at various interfaces. However, developing such tools requires validation by the experimental data. These experimental data are scarce because few physical methods can simultaneously distinguish chiral folding of the biopolymers, separate signals of interfaces from the overwhelming background of bulk solvent, and differentiate water in hydration shells of the polymers from water elsewhere.We recently illustrated these very capacities of chirality-sensitive vibrational sum frequency generation spectroscopy (chiral SFG). While chiral SFG theory dictates that the method is surface-specific under the condition of electronic nonresonance, we show the method can distinguish chiral folding of proteins and DNA and probe water structures in the first hydration shell of proteins at interfaces. Using amide I signals, we observe protein folding into ß-sheets without background signals from α-helices and disordered structures at interfaces, thereby demonstrating the effect of 2D crowding on protein folding. Also, chiral SFG signals of C-H stretches are silent from single-stranded DNA, but prominent for canonical antiparallel duplexes as well as noncanonical parallel duplexes at interfaces, allowing for sensing DNA secondary structures and hybridization. In establishing chiral SFG for detecting protein hydration structures, we observe an H218O isotopic shift that reveals water contribution to the chiral SFG spectra. Additionally, the phase of the O-H stretching bands flips when the protein chirality is switched from L to D. These experimental results agree with our simulated chiral SFG spectra of water hydrating the ß-sheet protein at the vacuum-water interface. The simulations further reveal that over 90% of the total chiral SFG signal comes from water in the first hydration shell. We conclude that the chiral SFG signals originate from achiral water molecules that assemble around the protein into a chiral supramolecular structure with chirality transferred from the protein. As water O-H stretches can reveal hydrogen-bonding interactions, chiral SFG shows promise in probing the structures and dynamics of water-biopolymer interactions at interfaces. Altogether, our work has created an experimental and computational framework for chiral SFG to elucidate biological functions at interfaces, setting the stage for probing the intricate chemical interplay of chirality, water, and interfaces.

Mirror-image antiparallel ß-sheets organize water molecules into superstructures of opposite chirality.

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 ß-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 ß-sheet. Therefore, we can conclude that the (d-) antiparallel ß-sheet organizes water solvent into a chiral supermolecular structure with opposite handedness relative to that of the (l-) antiparallel ß-sheet. We use molecular dynamics to characterize the chiral water superstructure at atomic resolution. The results show that the macroscopic chirality of antiparallel ß-sheets breaks the symmetry of assemblies of surrounding water molecules. We also calculate the chiral SFG response of water surrounding (l-) and (d-) LK7ß 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.

Chiral Sum Frequency Generation Spectroscopy Detects Double-Helix DNA at Interfaces.

Many DNA-based technologies involve the immobilization of DNA and therefore require a fundamental understanding of the DNA structure-function relationship at interfaces. We present three immobilization methods compatible with chiral sum frequency generation (SFG) spectroscopy at interfaces. They are the "anchor" method for covalently attaching DNA on a glass surface, the "island" method for dropcasting DNA on solid substrates, and the "buoy" method using a hydrocarbon moiety for localizing DNA at the air-water interface. Although SFG was previously used to probe DNA, the chiral and achiral SFG responses of single-stranded and double-stranded DNA have not been compared systemically. Using the three immobilization methods, we obtain the achiral and chiral C-H stretching spectra. The results introduce four potential applications of chiral SFG. First, chiral SFG gives null response from single-stranded DNA but prominent signals from double-stranded DNA, providing a simple binary readout for label-free detection of DNA hybridization. Second, with heterodyne detection, chiral SFG gives an opposite-signed spectral response useful for distinguishing native (D-) right-handed double helix from non-native (L-) left-handed double helix. Third, chiral SFG captures the aromatic C-H stretching modes of nucleobases that emerge upon hybridization, revealing the power of chiral SFG to probe highly localized molecular structures within DNA. Finally, chiral SFG is sensitive to macroscopic chirality but not local chiral centers and thus can detect not only canonical antiparallel double helix but also other DNA secondary structures, such as a poly-adenine parallel double helix. Our work benchmarks the SFG responses of DNA immobilized by the three distinct methods, building a basis for new chiral SFG applications to solve fundamental and biotechnological problems.

Collaboration between experiment and theory in solar fuels research.

As the challenges in science increase in scope and interdisciplinarity, collaboration becomes increasingly important. Our groups have maintained close collaborations for solar fuels research over the past decade. Based on this experience, we discuss strategies for collaboration between experiment and theory including facilitation of effective communication and navigation of problems that arise. These strategies are illustrated by case studies of collaborative efforts in solar fuels research pertaining to interfacial electron transfer in dye-sensitized metal oxides and the design and mechanism of water-oxidation catalysts.

Design of an Electrostatic Frequency Map for the NH Stretch of the Protein Backbone and Application to Chiral Sum Frequency Generation Spectroscopy.

We develop an electrostatic map for the vibrational NH stretch (amide A) of the protein backbone with a focus on vibrational chiral sum frequency generation spectroscopy (chiral SFG). Chiral SFG has been used to characterize protein secondary structure at interfaces using the NH stretch and to investigate chiral water superstructures around proteins using the OH stretch. Interpretation of spectra has been complicated because the NH stretch and OH stretch overlap spectrally. Although an electrostatic map for water OH developed by Skinner and co-workers was used previously to calculate the chiral SFG response of water structures around proteins, a map for protein NH that is directly responsive to biological complexity has yet to be developed. Here, we develop such a map, linking the local electric field to vibrational frequencies and transition dipoles. We apply the map to two protein systems and achieve much better agreement with experiment than was possible in our previous studies. We show that couplings between NH and OH vibrations are crucial to the line shape, which informs the interpretation of chiral SFG spectra, and that the chiral NH stretch response is sensitive to small differences in structure. This work increases the utility of the NH stretch in biomolecular spectroscopy.

Detecting the First Hydration Shell Structure around Biomolecules at Interfaces.

Understanding the role of water in biological processes remains a central challenge in the life sciences. Water structures in hydration shells of biomolecules are difficult to study in situ due to overwhelming background from aqueous environments. Biological interfaces introduce additional complexity because biomolecular hydration differs at interfaces compared to bulk solution. Here, we perform experimental and computational studies of chiral sum frequency generation (chiral SFG) spectroscopy to probe chirality transfer from a protein to the surrounding water molecules. This work reveals that chiral SFG probes the first hydration shell around the protein almost exclusively. We explain the selectivity to the first hydration shell in terms of the asymmetry induced by the protein structure and specific protein-water hydrogen-bonding interactions. This work establishes chiral SFG as a powerful technique for studying hydration shell structures around biomolecules at interfaces, presenting new possibilities to address grand research challenges in biology, including the molecular origins of life.

Simulation of the Chiral Sum Frequency Generation Response of Supramolecular Structures Requires Vibrational Couplings.

Chiral vibrational sum frequency generation (SFG) spectroscopy probes the structure of the solvation shell around chiral macromolecules. The dominant theoretical framework for understanding the origin of chiral SFG signals is based on the analysis of molecular symmetry, which assumes no interaction between molecules. However, water contains strong intermolecular interactions that significantly affect its properties. Here, the role of intermolecular vibrational coupling in the chiral SFG response of the O-H stretch of water surrounding an antiparallel ß-sheet at the vacuum-water interface is investigated. Both intramolecular and intermolecular couplings between O-H groups are required to simulate the full lineshape of the chiral SFG signal. This dependence is also observed for a chiral water dimer, illustrating that this phenomenon is not specific to larger systems. We also find that a dimer of C3v molecules predicted to be chirally SFG-inactive by the symmetry-based theory can generate a chiral SFG signal when intermolecular couplings are considered, suggesting that even highly symmetric solvent molecules may produce chiral SFG signals when interacting with a chiral solute. The consideration of intermolecular couplings extends the prevailing theory of the chiral SFG response to structures larger than individual molecules and provides guidelines for future modeling.

Chiral Water Superstructures around Antiparallel ß-Sheets Observed by Chiral Vibrational Sum Frequency Generation Spectroscopy.

Hydration modulates every aspect of protein structure and function. However, studying water structures in hydration shells remains challenging mostly due to overwhelming background from bulk water. We used vibrational sum frequency generation (SFG) spectroscopy to characterize hydrated films of an antiparallel ß-sheet peptide (LK7ß) adsorbed on glass slides. The hydrated films give chiral SFG response from water only when the peptide self-assembles into antiparallel ß-sheets. Experiments of isotopic labeling, isotopic dilution of water, and H2O-D2O exchange kinetics corroborate the assignments of the chiral SFG response to water stretching modes. Because individual water molecules are achiral, the chiral SFG response indicates formation of chiral superstructures of water around the antiparallel ß-sheet, implying that a protein secondary structure can imprint its chirality onto the surrounding water. This result demonstrates chiral SFG spectroscopy as a promising tool for probing water structures in protein hydration and addressing fundamental questions of protein structure-function.

Chiral Inversion of Amino Acids in Antiparallel ß-Sheets at Interfaces Probed by Vibrational Sum Frequency Generation Spectroscopy.

A parallel study of protein variants with all (l-), all (d-), or mixed (l-)/(d-) amino acids can be used to assess how backbone architecture versus side chain identity determines protein structure. Here, we investigate the secondary structure and side chain orientation dynamics of the antiparallel ß-sheet peptide LK7ß (Ac-Leu-Lys-Leu-Lys-Leu-Lys-Leu-NH2) composed of all (l-), all (d-), or alternating (l-Leu)/(d-Lys) amino acids. Using interface-selective vibrational sum frequency generation spectroscopy (VSFG), we observe that the alternating (l-)/(d-) peptide lacks a resonant C-H stretching mode compared to the (l-) and (d-) variants and does not form antiparallel ß-sheets. We rationalize our observations on the basis of density functional theory calculations and molecular dynamics (MD) simulations of LK7ß at the air-water interface. Irrespective of the handedness of the amino acids, leucine side chains prefer to orient toward the hydrophobic air phase while lysine side chains prefer the hydrophilic water phase. These preferences dictate the backbone configuration of LK7ß and thereby the folding of the peptide. Our MD simulations show that the preferred side chain orientations can force the backbone of a single strand of (l-) LK7ß at the air-water interface to adopt ß-sheet Ramachandran angles. However, denaturation of the ß-sheets at pH = 2 results in a negligible chiral VSFG amide I response. The combined computational and experimental results lend critical support to the theory that a chiral VSFG response requires macroscopic chirality, such as in ß-sheets. Our results can guide expectations about the VSFG optical responses of proteins and should improve understanding of how amino acid chirality modulates the structure and function of natural and de novo proteins at biological interfaces.