Strengthening Aqueous Electrolytes without Strengthening Water.

Aqueous electrolytes typically suffer from poor electrochemical stability; however, eutectic aqueous solutions-25 wt.% LiCl and 62 wt.% H3 PO4 -cooled to -78 °C exhibit a significantly widened stability window. Integrated experimental and simulation results reveal that, upon cooling, Li+ ions become less hydrated and pair up with Cl- , ice-like water clusters form, and H⋅⋅⋅Cl- bonding strengthens. Surprisingly, this low-temperature solvation structure does not strengthen water molecules' O-H bond, bucking the conventional wisdom that increasing water's stability requires stiffening the O-H covalent bond. We propose a more general mechanism for water's low temperature inertness in the electrolyte: less favorable solvation of OH- and H+ , the byproducts of hydrogen and oxygen evolution reactions. To showcase this stability, we demonstrate an aqueous Li-ion battery using LiMn2 O4 cathode and CuSe anode with a high energy density of 109 Wh/kg. These results highlight the potential of aqueous batteries for polar and extraterrestrial missions.

An Engineered Biliverdin-Compatible Cyanobacteriochrome Enables a Unique Ultrafast Reversible Photoswitching Pathway.

Cyanobacteriochromes (CBCRs) are promising optogenetic tools for their diverse absorption properties with a single compact cofactor-binding domain. We previously uncovered the ultrafast reversible photoswitching dynamics of a red/green photoreceptor AnPixJg2, which binds phycocyanobilin (PCB) that is unavailable in mammalian cells. Biliverdin (BV) is a mammalian cofactor with a similar structure to PCB but exhibits redder absorption. To improve the AnPixJg2 feasibility in mammalian applications, AnPixJg2_BV4 with only four mutations has been engineered to incorporate BV. Herein, we implemented femtosecond transient absorption (fs-TA) and ground state femtosecond stimulated Raman spectroscopy (GS-FSRS) to uncover transient electronic dynamics on molecular time scales and key structural motions responsible for the photoconversion of AnPixJg2_BV4 with PCB (Bpcb) and BV (Bbv) cofactors in comparison with the parent AnPixJg2 (Apcb). Bpcb adopts the same photoconversion scheme as Apcb, while BV4 mutations create a less bulky environment around the cofactor D ring that promotes a faster twist. The engineered Bbv employs a reversible clockwise/counterclockwise photoswitching that requires a two-step twist on ~5 and 35 picosecond (ps) time scales. The primary forward Pfr → Po transition displays equal amplitude weights between the two processes before reaching a conical intersection. In contrast, the primary reverse Po → Pfr transition shows a 2:1 weight ratio of the ~35 ps over 5 ps component, implying notable changes to the D-ring-twisting pathway. Moreover, we performed pre-resonance GS-FSRS and quantum calculations to identify the Bbv vibrational marker bands at ~659,797, and 1225 cm-1. These modes reveal a stronger H-bonding network around the BV cofactor A ring with BV4 mutations, corroborating the D-ring-dominant reversible photoswitching pathway in the excited state. Implementation of BV4 mutations in other PCB-binding GAF domains like AnPixJg4, AM1_1870g3, and NpF2164g5 could promote similar efficient reversible photoswitching for more directional bioimaging and optogenetic applications, and inspire other bioengineering advances.

Developing Bright Green Fluorescent Protein (GFP)-like Fluorogens for Live-Cell Imaging with Nonpolar Protein-Chromophore Interactions.

Fluorescence-activating proteins (FAPs) that bind a chromophore and activate its fluorescence have gained popularity in bioimaging. The fluorescence-activating and absorption-shifting tag (FAST) is a light-weight FAP that enables fast reversible fluorogen binding, thus advancing multiplex and super-resolution imaging. However, the rational design of FAST-specific fluorogens with large fluorescence enhancement (FE) remains challenging. Herein, a new fluorogen directly engineered from green fluorescent protein (GFP) chromophore by a unique double-donor-one-acceptor strategy, which exhibits an over 550-fold FE upon FAST binding and a high extinction coefficient of approximately 100,000 M-1 cm-1 , is reported. Correlation analysis of the excited state nonradiative decay rates and environmental factors reveal that the large FE is caused by nonpolar protein-fluorogen interactions. Our deep insights into structure-function relationships could guide the rational design of bright fluorogens for live-cell imaging with extended spectral properties such as redder emissions.

Transient electronic and vibrational signatures during reversible photoswitching of a cyanobacteriochrome photoreceptor.

Cyanobacteriochromes (CBCRs) are an emerging class of photoreceptors that are distant relatives of the phytochromes family. Unlike phytochromes, CBCRs have gained popularity in optogenetics due to their highly diverse spectral properties spanning the UV to near-IR region and only needing a single compact binding domain. AnPixJg2 is a CBCR that can reversibly photoswitch between its red-absorbing (15ZPr) and green-absorbing (15EPg) forms of the phycocyanobilin (PCB) cofactor. To reveal primary events of photoconversion, we implemented femtosecond transient absorption spectroscopy with a homemade LED box and a miniature peristaltic pump flow cell to track transient electronic responses of the photoexcited AnPixJg2 on molecular time scales. The 525 nm laser-induced Pg-to-Pr reverse conversion exhibits a ~3 ps excited-state lifetime before reaching the conical intersection (CI) and undergoing further relaxation on the 30 ps time scale to generate a long-lived Lumi-G ground state intermediate en route to Pr. The 650 nm laser-induced Pr-to-Pg forward conversion is less efficient than reverse conversion, showing a longer-lived excited state which requires two steps with ~13 and 217 ps time constants to enter the CI region. Furthermore, using a tunable ps Raman pump with broadband Raman probe on both the Stokes and anti-Stokes sides, we collected the pre-resonance ground-state femtosecond stimulated Raman spectroscopy (GS-FSRS) data with mode assignments aided by quantum calculations. Key vibrational marker bands at ~850, 1050, 1615, and 1649 cm-1 of the Pr conformer exhibit a notable blueshift to those of the Pg conformer inside AnPixJg2, reflecting the PCB chromophore terminal D (major) and A (minor) ring twist along the primary photoswitching reaction coordinate. This integrated ultrafast spectroscopy and computational platform has the potential to elucidate photochemistry and photophysics of more CBCRs and photoactive proteins in general, providing the highly desirable mechanistic insights to facilitate the rational design of functional molecular sensors and devices.

Watching an Engineered Calcium Biosensor Glow: Altered Reaction Pathways before Emission.

Biosensors have become an indispensable tool set in life sciences. Among them, fluorescent protein-based biosensors have great biocompatibility and tunable emission properties but their development is largely on trial and error. To facilitate a rational design, we implement tunable femtosecond stimulated Raman spectroscopy, aided by transient absorption and quantum calculations, to elucidate the working mechanisms of a single-site Pro377Arg mutant of an emission ratiometric Ca2+ biosensor based on a green fluorescent protein-calmodulin complex. Comparisons with the parent protein and the Ca2+-free/bound states unveil more structural inhomogeneity yet an overall faster excited-state proton-transfer (ESPT) reaction inside the Ca2+-bound biosensor. The correlated photoreactant and photoproduct vibrational modes in the excited state reveal more chromophore twisting and trapping in the Ca2+-bound state during ESPT and the largely conserved chromophore dynamics in the Ca2+-free state from parent protein. The uncovered structural dynamics insights throughout an ESPT reaction inside a calcium biosensor provide important design principles in maintaining a hydrophilic, less compact, and more homogeneous environment with directional H-bonding (from the chromophore to surrounding protein residues) via bioengineering methods to improve the ESPT efficiency and quantum yield while maintaining photostability.

nNOS immunoreactivity co-localizes with GABAergic and cholinergic neurons, and associates with ß-endorphinergic and met-enkephalinergic opioidergic fibers in rostral ventromedial medulla and A5 of the mouse.

The present study examined the co-expression of neuronal nitric oxide synthase (nNOS) in the rostral ventromedial medulla (RVM) and A5 regions of the mouse brainstem within several neurochemical populations involved in nociceptive modulation. Double immunohistochemical methods showed that nNOS+ neurons do not co-localize with serotonergic neurons within any of these regions. Within the RVM, the nuclei raphe magnus and gigantocellularis contain a population of nNOS+/GAD67+ neurons, and within the paragigantocellularis lateralis, there is a smaller population of nNOS+/CHAT+ neurons. Further, nNOS+ neurons overlap the region of expression of ß-endorphinergic and met-enkephalinergic fibers within the RVM. No co-labeling was found within the A5 for any of these populations. These findings suggest that pain-modulatory serotonergic neurons within the brainstem do not directly produce nitric oxide (NO). Rather, NO-producing neurons within the RVM belong to GABAergic and cholinergic cell populations, and are in a position to modulate or be modulated by local opioidergic neurons.

Tuning calcium biosensors with a single-site mutation: structural dynamics insights from femtosecond Raman spectroscopy.

Fluorescent protein biosensors are popular reporters for biological processes and life sciences, but their fundamental working mechanisms remain unclear. To characterize the functional fluorescence events on their native timescales, we implemented wavelength-tunable femtosecond stimulated Raman spectroscopy (FSRS) to shed light on a blue-green emission-ratiometric fluorescent protein based Ca2+ biosensor with a single Pro377Arg mutation. The transient Raman modes of the embedded chromophore from ca. 1000-1650 cm-1 exhibit characteristic intensity and frequency dynamics which infer the underlying atomic motions and photochemical reaction stages. Our experimental study reveals the hidden structural inhomogeneity of the protein local environment upon Ca2+ binding with the mutated arginine residue trapping multiple chromophore subpopulations, which manifest distinct time constants of ∼16 and 90 ps for excited state proton transfer (ESPT) following 400 nm photoexcitation. The altered ESPT reaction pathways and emission properties of the Ca2+ biosensor represent the foundational step of rationally designing advanced fluorescent protein biosensors to tune their functionalities by site-specifically altering the local environment (e.g., the active site) of the embedded chromophore.