Bacterial metabolism of bile acids promotes generation of peripheral regulatory T cells.

Intestinal health relies on the immunosuppressive activity of CD4+ regulatory T (Treg) cells1. Expression of the transcription factor Foxp3 defines this lineage, and can be induced extrathymically by dietary or commensal-derived antigens in a process assisted by a Foxp3 enhancer known as conserved non-coding sequence 1 (CNS1)2-4. Products of microbial fermentation including butyrate facilitate the generation of peripherally induced Treg (pTreg) cells5-7, indicating that metabolites shape the composition of the colonic immune cell population. In addition to dietary components, bacteria modify host-derived molecules, generating a number of biologically active substances. This is epitomized by the bacterial transformation of bile acids, which creates a complex pool of steroids8 with a range of physiological functions9. Here we screened the major species of deconjugated bile acids for their ability to potentiate the differentiation of pTreg cells. We found that the secondary bile acid 3ß-hydroxydeoxycholic acid (isoDCA) increased Foxp3 induction by acting on dendritic cells (DCs) to diminish their immunostimulatory properties. Ablating one receptor, the farnesoid X receptor, in DCs enhanced the generation of Treg cells and imposed a transcriptional profile similar to that induced by isoDCA, suggesting an interaction between this bile acid and nuclear receptor. To investigate isoDCA in vivo, we took a synthetic biology approach and designed minimal microbial consortia containing engineered Bacteroides strains. IsoDCA-producing consortia increased the number of colonic RORγt-expressing Treg cells in a CNS1-dependent manner, suggesting enhanced extrathymic differentiation.

Kinetic model for reversible radical transfer in ribonucleotide reductase.

The enzyme ribonucleotide reductase (RNR), which catalyzes the reduction of ribonucleotides to deoxynucleotides, is vital for DNA synthesis, replication, and repair in all living organisms. Its mechanism requires long-range radical translocation over ∼32 Šthrough two protein subunits and the intervening aqueous interface. Herein, a kinetic model is designed to describe reversible radical transfer in Escherichia coli RNR. This model is based on experimentally studied photoRNR systems that allow the photochemical injection of a radical at a specific tyrosine residue, Y356, using a photosensitizer. The radical then transfers across the interface to another tyrosine residue, Y731, and continues until it reaches a cysteine residue, C439, which is primed for catalysis. This kinetic model includes radical injection, an off-pathway sink, radical transfer between pairs of residues along the pathway, and the conformational flipping motion of Y731 at the interface. Most of the input rate constants for this kinetic model are obtained from previous experimental measurements and quantum mechanical/molecular mechanical free-energy simulations. Ranges for the rate constants corresponding to radical transfer across the interface are determined by fitting to the experimentally measured Y356 radical decay times in photoRNR systems. This kinetic model illuminates the time evolution of radical transport along the tyrosine and cysteine residues following radical injection. Further analysis identifies the individual rate constants that may be tuned to alter the timescale and probability of the injected radical reaching C439. The insights gained from this kinetic model are relevant to biochemical understanding and protein-engineering efforts with potential pharmacological implications.

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.

Theoretical basis for interpreting heterodyne chirality-selective sum frequency generation spectra of water.

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 ß-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.

Beyond the "spine of hydration": Chiral SFG spectroscopy detects DNA first hydration shell and base pair structures.

Experimental methods capable of selectively probing water at the DNA minor groove, major groove, and phosphate backbone are crucial for understanding how hydration influences DNA structure and function. Chiral-selective sum frequency generation spectroscopy (chiral SFG) is unique among vibrational spectroscopies because it can selectively probe water molecules that form chiral hydration structures around biomolecules. However, interpreting chiral SFG spectra is challenging since both water and the biomolecule can produce chiral SFG signals. Here, we combine experiment and computation to establish a theoretical framework for the rigorous interpretation of chiral SFG spectra of DNA. We demonstrate that chiral SFG detects the N-H stretch of DNA base pairs and the O-H stretch of water, exclusively probing water molecules in the DNA first hydration shell. Our analysis reveals that DNA transfers chirality to water molecules only within the first hydration shell, so they can be probed by chiral SFG spectroscopy. Beyond the first hydration shell, the electric field-induced water structure is symmetric and, therefore, precludes chiral SFG response. Furthermore, we find that chiral SFG can differentiate chiral subpopulations of first hydration shell water molecules at the minor groove, major groove, and phosphate backbone. Our findings challenge the scientific perspective dominant for more than 40 years that the minor groove "spine of hydration" is the only chiral water structure surrounding the DNA double helix. By identifying the molecular origins of the DNA chiral SFG spectrum, we lay a robust experimental and theoretical foundation for applying chiral SFG to explore the chemical and biological physics of DNA hydration.

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.

Uncovering protein-protein interactions through a team-based undergraduate biochemistry course.

How can we provide fertile ground for students to simultaneously explore a breadth of foundational knowledge, develop cross-disciplinary problem-solving skills, gain resiliency, and learn to work as a member of a team? One way is to integrate original research in the context of an undergraduate biochemistry course. In this Community Page, we discuss the development and execution of an interdisciplinary and cross-departmental undergraduate biochemistry laboratory course. We present a template for how a similar course can be replicated at other institutions and provide pedagogical and research results from a sample module in which we challenged our students to study the binding interface between 2 important biosynthetic proteins. Finally, we address the community and invite others to join us in making a larger impact on undergraduate education and the field of biochemistry by coordinating efforts to integrate research and teaching across campuses.

Characterizing Interfaces by Voronoi Tessellation.

The chemistry of interfaces differs markedly from that of the bulk. Calculation of interfacial properties depends strongly on the definition of the interface, which can lead to ambiguous results that vary between studies. There is a need for a method that can explicitly define the interfaces and boundaries in molecular systems. Voronoi tessellation offers an attractive solution to this problem through its ability to determine neighbors among specified groups of atoms. Here we discuss three cases where Voronoi tessellation combined with modeling of vibrational sum frequency generation (SFG) spectroscopy yields relevant insights: the breakdown of the air-water interface into clear and intuitive molecular layers, the study of the hydration shell in biological systems, and the acceleration of difficult spectral calculations where intermolecular vibrational couplings dominate. The utility of Voronoi tessellation has broad applications that extend beyond any single type of spectroscopy or system.

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.