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Solution NMR is typically applied to biological systems with molecular weights < 40 kDa whereas magic-angle-spinning (MAS) solid-state NMR traditionally targets very large, oligomeric proteins and complexes exceeding 500 kDa in mass, including fibrils and crystalline protein preparations. Here, we propose that the gap between these size regimes can be filled by the approach presented that enables investigation of large, soluble and fully protonated proteins in the range of 40-140 kDa. As a key step, ultracentrifugation produces a highly concentrated, gel-like state, resembling a dense phase in spontaneous liquid-liquid phase separation (LLPS). By means of three examples, a Sulfolobus acidocaldarius bifurcating electron transfer flavoprotein (SaETF), tryptophan synthases from Salmonella typhimurium (StTS) and their dimeric β-subunits from Pyrococcus furiosus (PfTrpB), we show that such samples yield well-resolved proton-detected 2D and 3D NMR spectra at 100 kHz MAS without heterogeneous broadening, similar to diluted liquids. Herein, we provide practical guidance on centrifugation conditions and tools, sample behavior, and line widths expected. We demonstrate that the observed chemical shifts correspond to those obtained from µM/low mM solutions or crystalline samples, indicating structural integrity. Nitrogen line widths as low as 20-30 Hz are observed. The presented approach is advantageous for proteins or nucleic acids that cannot be deuterated due to the expression system used, or where relevant protons cannot be re-incorporated after expression in deuterated medium, and it circumvents crystallization. Importantly, it allows the use of low-glycerol buffers in dynamic nuclear polarization (DNP) NMR of proteins as demonstrated with the cyanobacterial phytochrome Cph1., (© 2024. The Author(s).)

By combining the porosity of crystalline metal-organic frameworks (MOFs) with the unique processability of the liquid state, melt-quenched MOF glasses offer exciting opportunities for molecular separation. However, progress in this field is limited by two factors. Firstly, only very few MOFs melt at elevated temperatures and transform into stable glasses upon cooling the corresponding MOF liquid. Secondly, the MOF glasses obtained thus far feature only very small porosities and very small pore sizes. Here, we demonstrate solvent-assisted linker exchange (SALE) as a versatile method to prepare highly porous melt-quenched MOF glasses from the canonical ZIF-8. Two additional organic linkers are incorporated into the non-meltable ZIF-8, yielding high-entropy, linker-exchanged ZIF-8 derivatives undergoing crystal-to-liquid-to-glass phase transitions by thermal treatment. The ZIF-8 glasses demonstrate specific pore volumes of about 0.2 cm 3 g -1 , adsorb large amounts of technologically relevant C 3 and C 4 hydrocarbons, and feature high kinetic sorption selectivities for the separation of propylene from propane., (© 2024. The Author(s).)

Cholesterol oxidases (ChOxes) are enzymes that catalyze the oxidation of cholesterol to cholest-4-en-3-one. These enzymes find wide applications across various diagnostic and industrial settings. In addition, as a pathogenic factor of several bacteria, they have significant clinical implications. The current classification system for ChOxes is based on the type of bond connecting FAD to the apoenzyme, which does not adequately illustrate the enzymatic and structural characteristics of these proteins. In this study, we have adopted an integrative approach, combining evolutionary analysis, classic enzymatic techniques and computational approaches, to elucidate the distinct features of four various ChOxes from Rhodococcus sp. (RCO), Cromobacterium sp. (CCO), Pseudomonas aeruginosa (PCO) and Burkhoderia cepacia (BCO). Comparative and evolutionary analysis of substrate-binding domain (SBD) and FAD-binding domain (FBD) helped to reveal the origin of ChOxes. We discovered that all forms of ChOxes had a common ancestor and that the structural differences evolved later during divergence. Further examination of amino acid variations revealed SBD as a more variable compared to FBD independently of FAD coupling mechanism. Revealed differences in amino acid positions turned out to be critical in determining common for ChOxes properties and those that account for the individual differences in substrate specificity. A novel look with the help of chemical descriptors on found distinct features were sufficient to attempt an alternative classification system aimed at application approach. While univocal characteristics necessary to establish such a system remain elusive, we were able to demonstrate the substrate and protein features that explain the differences in substrate profile., Competing Interests: Declaration of competing interest The authors whose names are listed immediately below certify that they have NO affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers' bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript. The authors whose names are listed immediately below report the following details of affiliation or involvement in an organization or entity with a financial or non-financial interest in the subject matter or materials discussed in this manuscript. Please specify the nature of the conflict on a separate sheet of paper if the space below is inadequate. This statement is signed by all the authors to indicate agreement that the above information is true and correct (a photocopy of this form may be used if there are more than 10 authors)., (Copyright © 2023 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

With perdeuteration, solid-state NMR spectroscopy of large proteins suffers from incomplete amide-proton back-exchange. Using a 72 kDa micro-crystalline protein, we show that deuteration exclusively via deuterated amino acids, well-established in solution to suppress sidechain protonation without proton back-exchange obstacles, provides spectral resolution comparable to perdeuterated preparations at intermediate spinning frequencies.