David E. Hardy

Opsins are highly abundant retinal proteins in the membranes of photoheterotrophic bacteria. However, some microbial genomes encode an opsin but lack the gene for the final enzyme in retinal synthesis. To account for this paradox, we hypothesized that bacterial opsins play a role in membrane structure and/or biogenesis independent from their potential for light-driven signaling or proton pumping. After purifying actinorhodopsin from a cell-free expression system and from E. coli membranes upon overexpression, we demonstrated both in vitro and in silico that actinorhodopsin from Nanopelagicus ca. is a phospholipid scramblase, serving in its pentameric state as a retinal-independent phospholipid diffusion channel. Phospholipid headgroups move along a transbilayer path between actinorhodopsin protomers, to equilibrate lipid content in the inner and outer leaflets. Two profound activities, membrane biosynthesis and capture of light energy, are thus facilitated by one ancient bacterial polypeptide. Light-dependent activity and light-independent phospholipid scrambling are shared functions of eukaryotic, archaeal, and bacterial rhodopsins. Importance: Cells are surrounded by membranes which concentrate metabolites and protect cellular contents. Most biomembranes are phospholipid bilayers, in which the phospholipids of each leaflet orient their greasy tails inward and polar groups outward. Bilayer biogenesis depends on phospholipids synthesized on the cytofacial side of the membrane reorienting to the extracellular membrane leaflet. This reorientation requires proteins, termed scramblases, and it was shown that rhodopsins –– 7-helix photoactive membrane proteins bound to the cofactor retinal –– from organisms as widely divergent as mammals and archaea possess scramblase activity. Now we conclusively demonstrate using purified proteins in laboratory membranes as well as computational approaches, that bacterial rhodopsins are also phospholipid scramblases. This work is important because it highlights a surprising commonality among bacteria, archaea and eukaryotes and because it shows that rhodopsins – ancient proteins found in the last universal common ancestor – manifest two seemingly unrelated biochemical functions in one protein.

The cold chain system poses a significant obstacle to equitable global access to safe and effective nucleic acid-based vaccines. There is, therefore, a critical need to eliminate or improve this vaccine coverage gap by minimizing the costly and labor-intensive cold chain process, thereby providing equitable access to these life-saving therapeutics. In this work, quantum chemical evaluation qualitatively compared the interactions of a choline chloride/trehalose-based deep eutectic solvent (DES) with model bases (adenine (A), guanine (G), cytosine (C), thiamine (T), and uracil (U)) and their base pairs AT, AU, and GC in nucleic acids. We found that unpaired purines primarily interacted with the DES through C-H···π and hydrogen bonding, leading to cage-like structures, while pyrimidines primarily engaged through hydrogen bonding. In DES/base pair complexes, the AT base pair weakened and partially lost planarity due to C-H···π interactions, while the AU base pair was disrupted by additional hydrogen bonding with Cl^-. Conversely, the GC base pair retained its structure with strengthened hydrogen bonding in the presence of DES. Aromaticity increased in unpaired bases and base pairs AT and AU due to multiple hydrogen bonds but decreased in the DES/GC complex dominated by C-H···π interactions. Charge transfer analysis showed that purines lost electron density upon interacting with DES, while pyrimidines gained electron density. Interestingly, the GC and AT base pairs that retained pairing lost electron density to DES, whereas the AU base pair gained electron density, indicating complex interactions. The thermochemistry indicated favorable interactions of DES with unpaired bases and base pairs. However, it also suggests that once the bases are unpaired, it is energetically expensive for them to pair again.

In this work, we have provided mechanistic insight into the addition of bromine to an allylic double bond of allylaryl derivatives using experimental and DFT-based electronic structure methods. The experimental yields indicate the influence of the functional group on the aryl ring on the ratio of 1,2-dibromo and 1,3-dibromo adducts formed in the reaction. The optimized geometry and the electron density maps of the allylaryls and their cationic intermediates from DFT simulations revealed that electron-rich aryl rings promoted formation of cationic spiro[2.5] intermediate II, whereas electron-poor aryl rings resulted in formation of bromonium intermediate I. It was observed that electron-rich allylaryls promoted the 1,2-shift of the aryl ring that resulted in bond formation between the carbon atom (C1) on the aryl ring and the central carbon atom (C3) in the allylic double bond and formed spiro[2.5] intermediate II, a trend which was confirmed by harmonic oscillator model of aromaticity index. Also, Wiberg bond order analysis is in good agreement with the experimental work. Thermochemical analysis indicates that smaller C1C3 distance resulted in favorable values for the difference in free energy change (Delta Delta G). The favorable Delta Delta G values are a result of higher electron density on the aryl ring, making it more nucleophilic toward C3 carbon and promoting 1,2-shift that led to formation of the spiro[2.5] intermediate. Thus, the underlying mechanism indicates that the electron-rich allylaryls promote the formation of 1,3-dibromo compounds through formation and stabilization of the spiro[2.5] intermediate II.

A fundamental challenge underlying the design principles of ionic liquids (ILs) entails a lack of understanding into how tailored properties arise from the molecular framework of the constituent ions. Herein, we present detailed analyses of novel functional ILs containing a triarylmethyl (trityl) motif. Combining an empirically driven molecular design, thermophysical analysis, X-ray crystallography, and computational modeling, we achieved an in-depth understanding of structure-property relationships, establishing a coherent correlation with distinct trends between the thermophysical properties and functional diversity of the compound library. We observe a coherent relationship between melting (T _m) and glass transition (T _g) temperatures and the location and type of chemical modification of the cation. Furthermore, there is an inverse correlation between the simulated dipole moment and the T _m/T _g of the salts. Specifically, chlorination of the ILs both reduces and reorients the dipole moment, a key property controlling intermolecular interactions, thus allowing for control over T _m/T _g values. The observed trends are particularly apparent when comparing the phase transitions and dipole moments, allowing for the development of predictive models. Ultimately, trends in structural features and characterized properties align with established studies in physicochemical relationships for ILs, underpinning the formation and stability of these new lipophilic, low-melting salts.