© 2020, The Author(s), under exclusive licence to Springer Nature Limited. The use of nitrogen fertilizers has been estimated to have supported 27% of the world's population over the past century. Urea (CO(NH2)2) is conventionally synthesized through two consecutive industrial processes, N2 + H2 → NH3 followed by NH3 + CO2 → urea. Both reactions operate under harsh conditions and consume more than 2% of the world's energy. Urea synthesis consumes approximately 80% of the NH3 produced globally. Here we directly coupled N2 and CO2 in H2O to produce urea under ambient conditions. The process was carried out using an electrocatalyst consisting of PdCu alloy nanoparticles on TiO2 nanosheets. This coupling reaction occurs through the formation of C-N bonds via the thermodynamically spontaneous reaction between *N=N* and CO. Products were identified and quantified using isotope labelling and the mechanism investigated using isotope-labelled operando synchrotron-radiation Fourier transform infrared spectroscopy. A high rate of urea formation of 3.36 mmol g-1 h-1 and corresponding Faradic efficiency of 8.92% were measured at-0.4 V versus reversible hydrogen electrode.

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Although over expression and 15N enrichment facilitate the observation of resonances from disordered proteins in Escherichia coli, 15N enrichment alone is insufficient for detecting most globular proteins. Here we explain this dichotomy and overcome the problem while extending the capability of in-cell NMR by using 19F labeled proteins. Resonances from small (~10 kDa) globular proteins containing the amino acid analog 3-fluoro-tyrosine can be observed in cells, but for larger proteins the 19F resonances are broadened beyond detection. Incorporating the amino acid analog trifluoromethyl-L-phenylalanine allows larger proteins (up to 100 kDa) to be observed in cells. We also show that site specific structural and dynamic information about both globular and disordered proteins can be obtained inside cells by using 19F NMR.

Despite increased attention, little is known about how the crowded intracellular environment affects basic phenomena like protein diffusion. Here, we use NMR to quantify the rotational and translational diffusion of a 7.4-kDa test protein, chymotrypsin inhibitor 2 (CI2), in solutions of glycerol, synthetic polymers, proteins, and cell lysates. As expected, translational diffusion and rotational diffusion decrease with increasing viscosity. In glycerol, for example, the decrease follows the Stokes-Einstein and Stokes-Einstein-Debye laws. Synthetic polymers cause negative deviation from the Stokes Laws and affect translation more than rotation. Surprisingly, however, protein crowders have the opposite effect, causing positive deviation and reducing rotational diffusion more than translational diffusion. Indeed, bulk proteins severely attenuate the rotational diffusion of CI2 in crowded protein solutions. Similarly, CI2 diffusion in cell lysates is comparable to its diffusion in crowded protein solutions, supporting the biological relevance of the results. The rotational attenuation is independent of the size and total charge of the crowding protein, suggesting that the effect is general. The difference between the behavior of synthetic polymers and protein crowders suggests that synthetic polymers may not be suitable mimics of the intracellular environment. NMR relaxation data reveal that the source of the difference between synthetic polymers and proteins is the presence of weak interactions between the proteins and CI2. In summary, weak but non-specific, non-covalent chemical interactions between proteins appear to fundamentally impact protein diffusion in cells. Keywords In-cell NMR; Macromolecular crowding; Protein diffusion; Weak interactionsProtein diffusion affects many aspects of cell biology, from metabolism to signal transduction. The intracellular environment, however, is complex and difficult to study directly. Most work is performed in solutions where the total protein concentration is less than 10 g/L. These dilute solutions give optimal signals, but may lack biological relevance. Macromolecules occupy up to 30% of a cell's volume and reach concentrations of 100 to 400 g/L.1 Such large volume occupancies affect protein stability,2 folding,3 , 4 and aggregation,5 but only recently has attention been directed to the effects of macromolecular Materials and Methods15 N-enriched CI2 was expressed and purified as described.2 , 19 Chicken lysozyme, chicken ovalbumin, bovine serum albumin (BSA), Ficoll 70 (Ficoll) and polyvinylpyrrolidone 40 (PVP) were purchased from Sigma-Aldrich and used without further purification. Viscosities were measured with a Viscolite 700 viscometer (Hydramotion Ltd., England). Glycerol, PVP and Ficoll were dissolved in 50 mM sodium acetate (pH 5.4). A more concentrated buffer was required for proteins crowders. Lysozyme, ovalbumin and BSA were dissolved in 200 mM sodium acetate (pH 5.4). E. coli LysatesCultures of strain BL21 (DE3) Gold (Stratagene) containing an empty pE...

Amantadine is known to block the M2 proton channel of the Influenza A virus. Here, we present a structure of the M2 trans-membrane domain blocked with amantadine, built using orientational constraints obtained from solid-state NMR polarization-inversion-spin-exchange-at-the-magic-angle experiments. The data indicates a kink in the monomer between two helical fragments having 20 degrees and 31 degrees tilt angles with respect to the membrane normal. This monomer structure is then used to construct a plausible model of the tetrameric amantadine-blocked M2 trans-membrane channel. The influence of amantadine binding through comparative cross polarization magic-angle spinning spectra was also observed. In addition, spectra are shown of the amantadine-resistant mutant, S31N, in the presence and absence of amantadine.

Long-lived phosphorescence at room temperature (RTP) from pure organic molecules is rare. Recent research reveals various crystalline organic molecules can realize RTP with lifetimes extending to the magnitude of second. There is little research on how molecular packing affecting RTP. Three compounds are designed with similar optical properties in solution, but tremendously different solid emission characteristics. By investigating the molecular packing arrangement in single crystals, it is found that the packing style of the compact face to face favors of long phosphorescence lifetime and high photoluminescence efficiency, with the lifetime up to 748 ms observed in the crystal of CPM ((9H-carbazol-9-yl)(phenyl)methanone). Theoretical calculation analysis also reveals this kind of packing style can remarkably reduce the singlet excited energy level and prompt electron communication between dimers. Surprisingly, CPM has two very similar single crystals, labeled as CPM and CPM-A, with almost identical crystal data, and the only difference is that molecules in CPM-A crystal take a little looser packing arrangement. X-ray diffraction and cross-polarization under magic spinning C NMR spectra double confirm that they are different crystals. Interestingly, CPM-A crystal shows negligible RTP compared to the CPM crystal, once again proving that the packing style is critical to the RTP property.

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The intracellular milieu is complex, heterogeneous and crowded-an environment vastly different from dilute solutions in which most biophysical studies are performed. The crowded cytoplasm excludes about a third of the volume available to macromolecules in dilute solution. This excluded volume is the sum of two parts: steric repulsions and chemical interactions, also called soft interactions. Until recently, most efforts to understand crowding have focused on steric repulsions. Here, we summarize the results and conclusions from recent studies on macromolecular crowding, emphasizing the contribution of soft interactions to the equilibrium thermodynamics of protein stability. Despite their non-specific and weak nature, the large number of soft interactions present under many crowded conditions can sometimes overcome the stabilizing steric, excluded volume effect.