© 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...