Recent Advances in Solar Thermal Electrochemical Process (STEP) for Carbon Neutral Products and High Value Nanocarbons.

Climate change represents one of the most important environmental issues of our time. Due to high levels of anthropogenic CO2 emissions, atmospheric CO2 has for the first time ever exceeded 415 ppm and has increased from 315 ppm in 1950. An annual increase in atmospheric CO2 of ∼2 ppm is equal to a net increase of ∼15.6 billion tons of CO2. The combustion of fossil fuels for electricity and transportation is still the main reason accounting for the CO2 accumulation. On the top of that, fossil fuels are widely used in our modern industry for the productions of indispensable social staples. For instance, the millennia old thermal reduction of iron ore by charcoal or baked coal (3C + 2Fe2O3 → 4Fe + 3CO2) continues as the main method for the production of iron. The artificial fertilizer ammonia boosts the global population and is mainly produced from the Haber-Bosch process, in which hydrogen is generated via steam reforming of methane (CH4 + 2H2O → 4H2 + CO2). Sequestration and diminution of CO2 require the development of a portfolio of technologies on (1) efficient and long-term harvesting of renewable energy, that is, solar, not only for electricity but also directly as the energy force in vital chemical processes, wherever possible, (2) carbon-neutral processes to replace current industrial processes that emit vast amounts of CO2, such as iron and ammonia production, and (3) new, low-cost technologies for CO2 capture and conversion with particular interests in the exploration of CO2 as the feedstock for fuels or other valuable chemicals and materials. To this end, we conducted some studies on the sustainable synthesis of ammonia and iron with net-zero CO2 emissions and large-scale CO2 capture and conversion into fuels and high value nanocarbon products via electrolysis in molten salt(s) with the introduction of the Solar Thermal Electrochemical Process (STEP). In STEP, solar UV-visible energy is focused on a photovoltaic device that generates the electricity to drive the electrolysis, while concurrently the solar thermal energy is focused on a second system to generate heat for the electrolysis cell. The utilization of the full spectrum of sunlight in STEP results in a higher solar energy efficiency than other solar conversion processes. STEP has been applied to conduct (1) CO2-free ammonia synthesis from nitrogen and water with the aid of nano-Fe2O3 in a molten hydroxide electrolyte, (2) CO2-free production of iron via electrochemical reduction of iron ore in molten carbonate, (3) CO2 capture and conversion into nanostructured carbon products as well as fuels in molten or mixed molten electrolytes, and (4) organic electrosynthesis of benzoic acid from benzene without overoxidizing into CO2. In this Account, we highlight some recent achievements in these topics and propose that using STEP is a highly efficient strategy for saving energy and, consequently, the environment. STEP is an ideal tool that can theoretically be applied to all endothermic reactions.

Selective inhibition of mutant isocitrate dehydrogenase 1 (IDH1) via disruption of a metal binding network by an allosteric small molecule.

Cancer-associated point mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) confer a neomorphic enzymatic activity: the reduction of α-ketoglutarate to d-2-hydroxyglutaric acid, which is proposed to act as an oncogenic metabolite by inducing hypermethylation of histones and DNA. Although selective inhibitors of mutant IDH1 and IDH2 have been identified and are currently under investigation as potential cancer therapeutics, the mechanistic basis for their selectivity is not yet well understood. A high throughput screen for selective inhibitors of IDH1 bearing the oncogenic mutation R132H identified compound 1, a bis-imidazole phenol that inhibits d-2-hydroxyglutaric acid production in cells. We investigated the mode of inhibition of compound 1 and a previously published IDH1 mutant inhibitor with a different chemical scaffold. Steady-state kinetics and biophysical studies show that both of these compounds selectively inhibit mutant IDH1 by binding to an allosteric site and that inhibition is competitive with respect to Mg(2+). A crystal structure of compound 1 complexed with R132H IDH1 indicates that the inhibitor binds at the dimer interface and makes direct contact with a residue involved in binding of the catalytically essential divalent cation. These results show that targeting a divalent cation binding residue can enable selective inhibition of mutant IDH1 and suggest that differences in magnesium binding between wild-type and mutant enzymes may contribute to the inhibitors' selectivity for the mutant enzyme.

One-Pot Synthesis of Carbon Nanofibers from CO2.

Carbon nanofibers, CNFs, due to their superior strength, conductivity, flexibility, and durability have great potential as a material resource but still have limited use due to the cost intensive complexities of their synthesis. Herein, we report the high-yield and scalable electrolytic conversion of atmospheric CO2 dissolved in molten carbonates into CNFs. It is demonstrated that the conversion of CO2 → CCNF + O2 can be driven by efficient solar, as well as conventional, energy at inexpensive steel or nickel electrodes. The structure is tuned by controlling the electrolysis conditions, such as the addition of trace transition metals to act as CNF nucleation sites, the addition of zinc as an initiator and the control of current density. A less expensive source of CNFs will facilitate its adoption as a societal resource, and using carbon dioxide as a reactant to generate a value added product such as CNFs provides impetus to consume this greenhouse gas to mitigate climate change.

Advances in understanding the mechanism and improved stability of the synthesis of ammonia from air and water in hydroxide suspensions of nanoscale Fe2O3.

We report a mechanism of electrochemical ammonia (NH3) production via an iron intermediate in which H2 and NH3 are cogenerated by different electron-transfer pathways. Solar thermal can contribute to the energy to drive this synthesis, resulting in a STEP, solar thermal electrochemical process, for NH3. Enhancements are presented to this carbon dioxide (CO2)-free synthesis, which uses suspensions of nano-Fe2O3 in high-temperature hydroxide electrolytes at nickel and Monel electrodes. In a 200 °C molten eutectic Na(0.5)K(0.5)OH electrolyte, the 3 Faraday efficiency per mole of synthesized NH3, η(NH3), increases with decreasing current density, and at j(electrolysis) = 200, 25, 2, and 0.7 mA cm(-2), η(NH3) = 1%, 7%, 37%, and 71%, respectively. At 200 mA cm(-2), over 90% of applied current drives H2, rather than NH3, formation. Lower temperature supports greater electrolyte hydration. At 105 °C in the hydrated Na(0.5)K(0.5)OH electrolyte, η(NH3) increases and then is observed to be highly stable at η(NH3) = 24(+2)%.

An mRNA-based platform for the delivery of pathogen-specific IgA into mucosal secretions.

Colonization of the gut and airways by pathogenic bacteria can lead to local tissue destruction and life-threatening systemic infections, especially in immunologically compromised individuals. Here, we describe an mRNA-based platform enabling delivery of pathogen-specific immunoglobulin A (IgA) monoclonal antibodies into mucosal secretions. The platform consists of synthetic mRNA encoding IgA heavy, light, and joining (J) chains, packaged in lipid nanoparticles (LNPs) that express glycosylated, dimeric IgA with functional activity in vitro and in vivo. Importantly, mRNA-derived IgA had a significantly greater serum half-life and a more native glycosylation profile in mice than did a recombinantly produced IgA. Expression of an mRNA encoded Salmonella-specific IgA in mice resulted in intestinal localization and limited Peyer's patch invasion. The same mRNA-LNP technology was used to express a Pseudomonas-specific IgA that protected from a lung challenge. Leveraging the mRNA antibody technology as a means to intercept bacterial pathogens at mucosal surfaces opens up avenues for prophylactic and therapeutic interventions.

Therapeutic enzyme engineering using a generative neural network.

Enhancing the potency of mRNA therapeutics is an important objective for treating rare diseases, since it may enable lower and less-frequent dosing. Enzyme engineering can increase potency of mRNA therapeutics by improving the expression, half-life, and catalytic efficiency of the mRNA-encoded enzymes. However, sequence space is incomprehensibly vast, and methods to map sequence to function (computationally or experimentally) are inaccurate or time-/labor-intensive. Here, we present a novel, broadly applicable engineering method that combines deep latent variable modelling of sequence co-evolution with automated protein library design and construction to rapidly identify metabolic enzyme variants that are both more thermally stable and more catalytically active. We apply this approach to improve the potency of ornithine transcarbamylase (OTC), a urea cycle enzyme for which loss of catalytic activity causes a rare but serious metabolic disease.

Functional equivalence of the nicotinic acetylcholine receptor transmitter binding sites in the open state.

The subunits of the muscle-type nicotinic acetylcholine receptor (AChR) are not uniformly oriented in the resting closed conformation: the two alpha subunits are rotated relative to its non-alpha subunits. In contrast, all the subunits overlay well with one another when agonist is bound to the AChR, suggesting that they are uniformly oriented in the open receptor. This gating-dependent increase in orientational uniformity due to rotation of the alpha subunits might affect the relative affinities of the two transmitter binding sites, making the two affinities dissimilar (functionally non-equivalent) in the initial ligand-bound closed state but similar (functionally equivalent) in the open state. To test this hypothesis, we measured single-channel activity of the alphaG153S gain-of-function mutant receptor evoked by choline, and estimated the resting closed-state and open-state affinities of the two transmitter binding sites. Both model-independent analyses and maximum-likelihood estimation of microscopic rate constants indicate that channel opening makes the binding sites' affinities more similar to each other. These results support the hypothesis that open-state affinities to the transmitter binding sites are primarily determined by the alpha subunits.

Binding Rate Screen - a high-throughput assay in soluble lysate for prioritizing protein expression constructs.

Identification of constructs suitable for the recombinant protein production pipeline is a bottleneck for structural genomics efforts, as most methods require purified proteins and/or are labor-intensive. Here, we present a novel high-throughput approach, Binding Rate Screen, that can alleviate this bottleneck by screening expression constructs in crude soluble lysate. This functional screen utilizes the frequently employed hexahistidine (His(6)) tag as a reporter, and measures its binding rate to an affinity matrix as a metric to reflect aggregation, concentration, and purifiability of the target protein. The constructs with the highest binding rates also exhibit high expression of soluble monomeric protein as judged by analytical size-exclusion chromatography. Constructs expressing variations of the target protein can be prioritized on a time scale of minutes, which is at least 10-100 times faster than any other technologies currently available.

Synchrotron protein footprinting supports substrate translocation by ClpA via ATP-induced movements of the D2 loop.

Synchrotron X-ray protein footprinting is used to study structural changes upon formation of the ClpA hexamer. Comparative solvent accessibilities between ClpA monomer and ClpA hexamer samples are in agreement throughout most of the sequence, with calculations based on two previously proposed hexameric models. The data differ substantially from the proposed models in two parts of the structure: the D1 sensor 1 domain and the D2 loop region. The results suggest that these two regions can access alternate conformations in which their solvent protection is greater than that in the structural models based on crystallographic data. In combination with previously reported structural data, the footprinting data provide support for a revised model in which the D2 loop contacts the D1 sensor 1 domain in the ATP-bound form of the complex. These data provide the first direct experimental support for the nucleotide-dependent D2 loop conformational change previously proposed to mediate substrate translocation.

Calcium metaborate induced thin walled carbon nanotube syntheses from CO2 by molten carbonate electrolysis.

An electrosynthesis is presented to transform the greenhouse gas CO2 into an unusually thin walled, smaller diameter morphology of Carbon Nanotubes (CNTs). The transformation occurs at high yield and coulombic efficiency of the 4-electron CO2 reduction in a molten carbonate electrolyte. The electrosynthesis is driven by an unexpected synergy between calcium and metaborate. In a pure molten lithium carbonate electrolyte, thicker walled CNTs (100-160 nm diameter) are synthesized during a 4 h CO2 electrolysis at 0.1 A cm-2. At this low current density, CO2 without pre-concentration is directly absorbed by the air (direct air capture) to renew and sustain the carbonate electrolyte. The addition of 2 wt% Li2O to the electrolyte produces thinner, highly uniform (50-80 nm diameter) walled CNTs, consisting of ~ 75 concentric, cylindrical graphene walls. The product is produced at high yield (the cathode product consists of > 98% CNTs). It had previously been demonstrated that the addition of 5-10 wt% lithium metaborate to the lithium carbonate electrolyte boron dopes the CNTs increasing their electrical conductivity tenfold, and that the addition of calcium carbonate to a molten lithium carbonate supports the electrosynthesis of thinner walled CNTs, but at low yield (only ~ 15% of the product are CNTs). Here it is shown that the same electrolysis conditions, but with the addition of 7.7 wt% calcium metaborate to lithium carbonate, produces unusually thin walled CNTs uniform (22-42 nm diameter) CNTs consisting of ~ 25 concentric, cylindrical graphene walls at a high yield of > 90% CNTs.

One pot facile transformation of CO2 to an unusual 3-D nano-scaffold morphology of carbon.

An electrosynthesis is presented to transform CO2 into an unusual nano and micron dimensioned morphology of carbon, termed Carbon Nano-Scaffold (CNS) with wide a range of high surface area graphene potential usages including batteries, supercapacitors, compression devices, electromagnetic wave shielding and sensors. Current CNS value is over $323 per milligram. The morphology consists of a series of asymmetric 20 to 100 nm thick flat multilayer graphene platelets 2 to 20 µm long orthogonally oriented in a 3D neoplasticism-like geometry, and appears distinct from the honeycomb, foam, or balsa wood cell structures previously attributed to carbon scaffolds. The CNS synthesis splits CO2 by electrolysis in molten carbonate and has a carbon negative footprint. It is observed that transition metal nucleated, high yield growth of carbon nanotubes (CNTs) is inhibited in electrolytes containing over 50 wt% of sodium or 30 wt% of potassium carbonate, or at electrolysis temperatures less than 700 °C. Here, it is found that a lower temperature of synthesis, lower concentrations of lithium carbonate, and higher current density promotes CNS growth while suppressing CNT growth. Electrolyte conditions of 50 wt% sodium carbonate relative to lithium carbonate at an electrolysis temperature of 670 °C produced over 80% of the CNS desired product at 85% faradaic efficiency with a Muntz brass cathode and an Inconel anode.

Polymer-based open-channel blockers for the acetylcholine receptor: the effect of spacer length on blockade kinetics.

Blocking open ion channels provides a promising way to modulate synaptic transmission. Using the muscle-type acetylcholine receptor (AChR) as a test system, we seek to develop blockers that have blockade kinetics tunable via structural modifications. Here we investigate whether the blockade kinetics can be modulated by specifying the length of a poly(ethylene glycol) (PEG) spacer incorporated into the blocker. Single-channel electrophysiological experiments show that simple bis(trimethylammonium) compounds ( 1a- 3) both activate the AChR and block the open channel. The blockade kinetics are found to depend on spacer length: both the association and dissociation rate constants decrease with increasing spacer length. The decrease in the association rate constant can be quantitatively explained by the entropic cost of polymer confinement in the transmembrane lumen, while the decrease in the dissociation rate constant is consistent with weak, additive noncovalent interactions between the channel and the spacer. These results provide useful insights into the future design of kinetically tunable open-channel blockers for the AChR.

Role of a conserved pore residue in the formation of a prehydrolytic high substrate affinity state in the AAA+ chaperone ClpA.

The AAA+ protease ClpAP, consisting of the ClpA chaperone and the ClpP protease, processively unfolds and translocates its substrates into its proteolytic core, where they are cleaved. Unfolding and efficient translocation require ATP-dependent conformational changes in ClpA's D2 loop, where the conserved GYVG motif resides. To explore the role of the essential tyrosine of this motif, we investigated how two mutations at this residue (Y540C and Y540A) affect the rate at which the enzyme processes unstructured substrates. The mutations decrease ClpA's ability to process unfolded or unstable protein substrates but have only mild effects on the rates of ATP hydrolysis or hydrolysis of small peptide substrates. The mutants' substrate binding properties were also characterized, using single molecule fluorescence microscopy. The single-molecule studies demonstrate that the conserved tyrosine is essential for the formation of the prehydrolytic, high substrate affinity conformation observed in wild-type ClpA. Together, the results support a model in which destabilization of the high substrate affinity conformation of ClpA makes translocation less efficient and uncouples it from ATP hydrolysis.

The ClpP N-terminus coordinates substrate access with protease active site reactivity.

Energy-dependent protein degradation machines, such as the Escherichia coli protease ClpAP, require regulated interactions between the ATPase component (ClpA) and the protease component (ClpP) for function. Recent studies indicate that the ClpP N-terminus is essential in these interactions, yet the dynamics of this region remain unclear. Here, we use synchrotron hydroxyl radical footprinting and kinetic studies to characterize functionally important conformational changes of the ClpP N-terminus. Footprinting experiments show that the ClpP N-terminus becomes more solvent-exposed upon interaction with ClpA. In the absence of ClpA, deletion of the ClpP N-terminus increases the initial degradation rate of large peptide substrates 5-15-fold. Unlike ClpAP, ClpPDeltaN exhibits a distinct slow phase of product formation that is eliminated by the addition of hydroxylamine, suggesting that truncation of the N-terminus leads to stabilization of the acyl-enzyme intermediate. These results indicate that (1) the ClpP N-terminus acts as a "gate" controlling substrate access to the active sites, (2) binding of ClpA opens this "gate", allowing substrate entry and formation of the acyl-enzyme intermediate, and (3) closing of the N-terminal "gate" stimulates acyl-enzyme hydrolysis.

ClpP hydrolyzes a protein substrate processively in the absence of the ClpA ATPase: mechanistic studies of ATP-independent proteolysis.

ATP-dependent proteases are processive, meaning that they degrade full-length proteins into small peptide products without releasing large intermediates along the reaction pathway. In the case of the bacterial ATP-dependent protease ClpAP, ATP hydrolysis by the ClpA component has been proposed to be required for processive proteolysis of full-length protein substrates. We present here data showing that in the absence of the ATPase subunit ClpA, the protease subunit ClpP can degrade full-length protein substrates processively, albeit at a greatly reduced rate. Moreover, the size distribution of peptide products from a ClpP-catalyzed digest is remarkably similar to the size distribution of products from a ClpAP-catalyzed digest. The ClpAP- and ClpP-generated peptide product size distributions are fitted well by a sum of multiple underlying Gaussian peaks with means at integral multiples of approximately 900 Da (7-8 amino acids). Our results are consistent with a mechanism in which ClpP controls product sizes by alternating between translocation in steps of 7-8 (+/-2-3) amino acid residues and proteolysis. On the structural and molecular level, the step size may be controlled by the spacing between the ClpP active sites, and processivity may be achieved by coupling peptide bond hydrolysis to the binding and release of substrate and products in the protease chamber.

An activity-based protein profiling probe for the nicotinic acetylcholine receptor.

Activity-based protein profiling (ABPP) has been used extensively to characterize the physiological functions of enzymes but has not yet been extended to ion channels. We have synthesized a state-dependent photoaffinity probe for the nicotinic acetylcholine receptor (nAChR) as a proof of concept for the development of ion channel directed ABPP probes. The candidate probe BPyneTEA comprises an nAChR binding moiety, a benzophenone moiety for photolabeling, and an alkyne moiety for biotinylation via "click chemistry". Single-molecule current measurements show that BPyneTEA blocks both the closed and open (i.e., nondesensitized) conformations of the nAChR with similar kinetics. In living cells, BPyneTEA photolabels the closed state selectively over the inactive desensitized state. BPyneTEA thus shows promise as a probe for nondesensitized nAChRs and may be useful in studying the molecular physiology of desensitization. The structure and reactivity of ion channel pores in general suggest that they will be a broadly useful target for ABPP probes.

Testing for violations of microscopic reversibility in ATP-sensitive potassium channel gating.

In pancreatic beta cells, insulin secretion is tightly controlled by the cells' metabolic state via the ATP-sensitive potassium (KATP) channel. ATP is a key mediator in this signaling process, where its role as an inhibitor of KATP channels has been extensively studied. Since the channel contains an ATPase as an accessory subunit, the possibility that ATP hydrolysis mediates KATP channel opening has also been proposed. However, a rigorous test of coupling between ATP hydrolysis and channel gating has not previously been performed. In the present work, we examine whether KATP channel gating obeys detailed balance in order to determine whether ATP hydrolysis is strongly coupled to the gating of the KATP channel. Single-channel records were obtained from inside-out patches of transiently transfected HEK-293 cells. Channel activity in membrane patches with exactly one channel shows no violations of microscopic reversibility. Although KATP channel gating shows long closed times on the time scale where ATP hydrolysis takes place, the time symmetry of channel gating indicates that it is not tightly coupled to ATP hydrolysis. This lack of coupling suggests that channel gating operates close to equilibrium; although detailed balance is not expected to hold for ATP hydrolysis, it still does so in channel gating. On the basis of these results, the function of the ATPase active site in channel gating may be to sense nucleotides by differential binding of ATP and ADP, rather than to drive a thermodynamically unfavorable conformational change.

Renewable highest capacity VB2/air energy storage.

The first renewable electrochemical energy system which stores more energy than gasoline is presented, and with an order of magnitude higher capacity than lithium-ion batteries, VB(2) opens a pathway towards electric vehicles with a viable driving range.

A Robust Multiplex Mass Spectrometric Assay for Screening Small-Molecule Inhibitors of CD73 with Diverse Inhibition Modalities.

CD73/Ecto-5'-nucleotidase is a membrane-tethered ecto-enzyme that works in tandem with CD39 to convert extracellular adenosine triphosphate (ATP) into adenosine. CD73 is highly expressed on various types of cancer cells and on infiltrating suppressive immune cells, leading to an elevated concentration of adenosine in the tumor microenvironment, which elicits a strong immunosuppressive effect. In preclinical studies, targeting CD73 with anti-CD73 antibody results in favorable antitumor effects. Despite initial studies using antibodies, inhibition of CD73 catalytic activity using small-molecule inhibitors may be more effective in lowering extracellular adenosine due to better tumor penetration and distribution. To screen small-molecule libraries, we explored multiple approaches, including colorimetric and fluorescent biochemical assays, and due to some limitations with these assays, we developed a mass spectrometry (MS)-based assay. Only the MS-based assay offers the sensitivity and dynamic range required for screening small-molecule libraries at a substrate concentration close to the Km value of substrate and for evaluating the mode of binding of screening hits. To achieve a throughput suitable for high-throughput screening (HTS), we developed a RapidFire-tandem mass spectrometry (RF-MS/MS)-based multiplex assay. This assay allowed a large diverse compound library to be screened at a speed of 1536 reactions per 40-50 min.

Dependence on the Pyrimidine Biosynthetic Enzyme DHODH Is a Synthetic Lethal Vulnerability in Mutant KRAS-Driven Cancers.

Activating KRAS mutations are major oncogenic drivers in multiple tumor types. Synthetic lethal screens have previously been used to identify targets critical for the survival of KRAS mutant cells, but their application to drug discovery has proven challenging, possibly due in part to a failure of monolayer cultures to model tumor biology. Here, we report the results of a high-throughput synthetic lethal screen for small molecules that selectively inhibit the growth of KRAS mutant cell lines in soft agar. Chemoproteomic profiling identifies the target of the most KRAS-selective chemical series as dihydroorotate dehydrogenase (DHODH). DHODH inhibition is shown to perturb multiple metabolic pathways. In vivo preclinical studies demonstrate strong antitumor activity upon DHODH inhibition in a pancreatic tumor xenograft model.