Search

Resonance assignments recently obtained on immobilized polypeptides and a membrane protein aggregate under Magic Angle Spinning are compared to random coil values in the liquid state. The resulting chemical shift differences (secondary chemical shifts) are evaluated in light of the backbone torsion angle ψ previously reported using X-ray crystallography. In all cases, a remarkable correlation is found suggesting that the concept of secondary chemical shifts, well established in the liquid state, can be of similar importance in the context of multiple-labelled polypeptides studied under MAS conditions. [ABSTRACT FROM AUTHOR]

One- and two-dimensional solid-state NMR experiments on a uniformly labeled intrinsic membrane-protein complex at ultra-high magnetic fields are presented. Two-dimensional backbone and side-chain correlations for a [U-13C,15N] labeled version of the LH2 light-harvesting complex indicate significant resolution at low temperatures and under Magic Angle Spinning. Tentative assignments of some of the observed correlations are presented and attributed to the α-helical segments of the protein, mostly found in the membrane interior. [ABSTRACT FROM AUTHOR]

Chédiak-Higashi-Steinbrinck syndrome (CHS) is a rare autosomal recessive disorder which is usually lethal in early childhood. Diagnostic hallmark is the occurrence of giant inclusion bodies in peripheral leukocytes and their bone marrow precursors. We report on a 27-year-old female patient who was admitted for treatment of a skin abscess. She recovered after intravenous antibiotic treatment and surgical incision. Hematological investigation was initiated because of a persisting neutropenia of 15%, with a leukocyte count initially in the normal range but subsequent leukopenia. Case history revealed recurrent skin infections from childhood on, regularly requiring surgical intervention. One year prior to admission a neuropathy had been diagnosed, while a partial albinism had been known for years. Microscopic examinations of peripheral blood and bone marrow aspirate smears were diagnostic for CHS. Additionally, a secondary antibody deficiency was found. Normalization of the white blood cell count, including the differential count, was observed following initiation of G-CSF treatment. Functional assessment of phagocytosis and oxidative burst activity of granulocytes revealed normal results before and after stimulation with G-CSF, however, natural killer cell activity was only weak, with slight improvement after G-CSF treatment in vivo. Cytogenetic analysis showed a normal female karyotype. Although the haploidentical brother of the patient may serve as an allogeneic stem cell donor, transplantation has been postponed because of further deterioration of her already existing CHS-specific neurological impairment. Nevertheless, while receiving G-CSF maintenance treatment our patient experienced no further infectious episodes within 6 months after diagnosis of CHS. [ABSTRACT FROM AUTHOR]

Examines through-bond connectives and through-space distances in solids by magic-angle-spinning nuclear magnetic resonance. Improved structure determination schemes; Employment of experimental techniques under high-resolution conditions; Use of magic-angle sample spinning.

Studies the crystal structure of anhydrous disodium hydrogen phosphate using solid-state NMR and powder X-ray diffraction techniques. Chemical shift and quadrupole interaction parameters; Consistency of the three inequivalent sodium sites with a P21/c crystal structure; Atomic coordinates.

We report magic angle spinning, dynamic nuclear polarization (DNP) experiments at magnetic fields of 9.4 T, 14.1 T, and 18.8 T using the narrow line polarizing agents 1,3-bisdiphenylene-2-phenylallyl (BDPA) dispersed in polystyrene, and sulfonated-BDPA (SA-BDPA) and trityl OX063 in glassy glycerol/water matrices. The ¹H DNP enhancement field profiles of the BDPA radicals exhibit a significant DNP Overhauser effect (OE) as well as a solid effect (SE) despite the fact that these samples are insulating solids. In contrast, trityl exhibits only a SE enhancement. Data suggest that the appearance of the OE is due to rather strong electron-nuclear hyperfine couplings present in BDPA and SA-BDPA, which are absent in trityl and perdeuterated BDPA (d21 -BDPA). In addition, and in contrast to other DNP mechanisms such as the solid effect or cross effect, the experimental data suggest that the OE in non-conducting solids scales favorably with magnetic field, increasing in magnitude in going from 5 T, to 9.4 T, to 14.1 T, and to 18.8 T. Simulations using a model two spin system consisting of an electron hyperfine coupled to a ¹H reproduce the essential features of the field profiles and indicate that the OE in these samples originates from the zero and double quantum cross relaxation induced by fluctuating hyperfine interactions between the intramolecular delocalized unpaired electrons and their neighboring nuclei, and that the size of these hyperfine couplings is crucial to the magnitude of the enhancements. Microwave power dependent studies show that the OE saturates at considerably lower power levels than the solid effect in the same samples. Our results provide new insights into the mechanism of the Overhauser effect, and also provide a new approach to perform DNP experiments in chemical, biophysical, and physical systems at high magnetic fields. [ABSTRACT FROM AUTHOR]

• Deactivation of ZrO 2 -promoted ZSM-5 catalyst extrudates in ex-situ CFP at bench scale. • Coking responsible for the decreased activity in cracking and deoxygenation. • Egg-shell spatial distribution of coke deposits within the catalyst extrudates. • Regeneration at 500 °C removes coke with no negative effect on catalytic properties. A major issue in the catalytic fast pyrolysis (CFP) of biomass is the rapid deactivation of the typically employed zeolite-based catalysts. Detailed understanding of the deactivation pathways and the type and location of coke deposits are essential for the further development of improved or new catalyst materials, including appropriate regeneration protocols. Such deactivation and regeneration studies focus almost invariably on small-scale CFP reactor units employing catalyst materials in powder form. In this study, we report the in-depth characterization of deactivated and regenerated ZrO 2 -promoted zeolite ZSM-5 catalyst extrudates after ex-situ CFP tests carried out in a bench scale reactor. The findings support that coking is the main reason for catalyst deactivation, i.e. for the observed decreased activity in cracking and deoxygenation. Post-mortem characterization by confocal fluorescence microscopy reveals an egg-shell spatial distribution of the coke deposits within the catalyst extrudates. These deposits are heavily poly-aromatic in nature. The majority of the coke build-up occurs in the first 20 min of the reaction and is formed on the strong Brønsted acid sites, which promote deep deoxygenation and cracking. With increasing time-on-stream, the coke deposition slows down, occurring now mainly on the external surface of the zeolite to generate a softer, i.e. more hydrogen-rich, coke on the ZrO 2 domains. The catalyst is readily regenerated via thermal oxidation in air, with optimal regeneration at 500 °C. This temperature removes all coke deposits, with no detrimental effect on the catalyst's structural, textural and acid (type and strength) properties. [ABSTRACT FROM AUTHOR]

We report solid-state Nuclear Magnetic Resonance (ssNMR) studies on amyloid-like protein complexes formed by DIX domains that mediate key protein interactions in the Wnt signalling pathway. Our results provide insight into the 3D fold of the self-associated Axin-DIX domain and identify a potential lipid cofactor. [ABSTRACT FROM AUTHOR]

Instruct-ERIC, "the European Research Infrastructure Consortium for Structural biology research," is a pan-European distributed research infrastructure making high-end technologies and methods in structural biology available to users. Here, we describe the current state-of-the-art of integrated structural biology and discuss potential future scientific developments as an impulse for the scientific community, many of which are located in Europe and are associated with Instruct. We reflect on where to focus scientific and technological initiatives within the distributed Instruct research infrastructure. This review does not intend to make recommendations on funding requirements or initiatives directly, neither at the national nor the European level. However, it addresses future challenges and opportunities for the field, and foresees the need for a stronger coordination within the European and international research field of integrated structural biology to be able to respond timely to thematic topics that are often prioritized by calls for funding addressing societal needs., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The ability to adapt to osmotically diverse and fluctuating environments is critical to the survival and resilience of bacteria that colonize the human gut and urinary tract. Environmental stress often provides cross-protection against other challenges and increases antibiotic tolerance of bacteria. Thus, it is critical to understand how E. coli and other microbes survive and adapt to stress conditions. The osmotically inducible protein Y (OsmY) is significantly upregulated in response to hypertonicity. Yet its function remains unknown for decades. We determined the solution structure and dynamics of OsmY by nuclear magnetic resonance spectroscopy, which revealed that the two Bacterial OsmY and Nodulation (BON) domains of the protein are flexibly linked under low- and high-salinity conditions. In-cell solid-state NMR further indicates that there are no gross structural changes in OsmY as a function of osmotic stress. Using cryo-electron and super-resolution fluorescence microscopy, we show that OsmY attenuates plasmolysis-induced structural changes in E. coli and improves the time to growth resumption after osmotic upshift. Structure-guided mutational and functional studies demonstrate that exposed hydrophobic residues in the BON1 domain are critical for the function of OsmY. We find no evidence for membrane interaction of the BON domains of OsmY, contrary to current assumptions. Instead, at high ionic strength, we observe an interaction with the water channel, AqpZ. Thus, OsmY does not play a simple structural role in E. coli but may influence a cascade of osmoregulatory functions of the cell., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Visualizing a protein's molecular motions has been a long standing topic of research in the biophysics community. Largely this has been done by exploiting nuclear magnetic resonance spectroscopy (NMR), and arguably no protein's molecular motions have been better characterized by NMR than that of ubiquitin (Ub), a 76 amino acid polypeptide essential in ubiquitination-a key regulatory system within cells. Herein, we discuss ubiquitin's conformational plasticity as visualized, at atomic resolution, by more than 35 years of NMR work. In our discussions we point out the differences between data acquired in vitro, ex vivo, as well as in vivo and stress the need to investigate Ub's conformational plasticity in more biologically representative backgrounds., (© 2024 The Author(s). ChemBioChem published by Wiley-VCH GmbH.)

Cross-linking, also called tanning, improves mechanical properties of leather and also increases its enzymatic and thermal stability. As a final product, leather has an ultimate tensile strength (σ) of 8-25 MPa and an elongation at break (ε) of >30 %. Mycelium-based materials are a sustainable alternative to leather. Here, the effect of cross-linkers was assessed on mechanical properties of Schizophyllum commune mycelium sheets . To this end, glutaraldehyde and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) were used as well as extracts of Ligustrum vulgare leaves , and bark of Acacia mearnsii and Caesalpinia spinosa. Untanned sheets had a σ of 7.8 MPa and an ε of 15.2 %, while the best overall combination of strength and elasticity was obtained with 0.1 % glutaraldehyde with a σ of 11.1 MPa and an ε of 14.6 %. Cross-linking also increased enzymatic stability and reduced mycelial water absorption but did not result in increased thermal stability. Fourier transform infrared spectroscopy (FTIR), 1D nuclear magnetic resonance spectroscopy (NMR), and amino acid analysis showed that glutaraldehyde bound both protein amino groups and polysaccharide hydroxyl groups by forming Schiff bases and acetals, respectively. Together, synthetic and vegetable cross-linkers can be used to obtain mycelium materials with leather-like tensile strength., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Antimicrobial resistance is a leading cause of mortality, calling for the development of new antibiotics. The fungal antibiotic plectasin is a eukaryotic host defence peptide that blocks bacterial cell wall synthesis. Here, using a combination of solid-state nuclear magnetic resonance, atomic force microscopy and activity assays, we show that plectasin uses a calcium-sensitive supramolecular killing mechanism. Efficient and selective binding of the target lipid II, a cell wall precursor with an irreplaceable pyrophosphate, is achieved by the oligomerization of plectasin into dense supra-structures that only form on bacterial membranes that comprise lipid II. Oligomerization and target binding of plectasin are interdependent and are enhanced by the coordination of calcium ions to plectasin's prominent anionic patch, causing allosteric changes that markedly improve the activity of the antibiotic. Structural knowledge of how host defence peptides impair cell wall synthesis will likely enable the development of superior drug candidates., (© 2024. The Author(s).)

Sensitivity enhanced dynamic nuclear polarization solid-state NMR is emerging as a powerful technique for probing the structural properties of conformationally homogenous and heterogenous biomolecular species irrespective of size at atomic resolution within their native environments. Herein we detail advancements that have made acquiring such data, specifically within the confines of intact bacterial and eukaryotic cell a reality and further discuss the type of structural information that can presently be garnered by the technique's exploitation. Subsequently, we discuss bottlenecks that have thus far curbed cellular DNP-ssNMR's broader adoption namely due a lack of sensitivity and spectral resolution. We also explore possible solutions ranging from utilization of new pulse sequences, design of better performing polarizing agents, and application of additional biochemical/ cell biological methodologies., (© 2024 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

Humins, (side-)products of the acid-catalysed dehydration of carbohydrates, will be produced in substantial quantities with the development of industrial biorefining processes. Most structural knowledge about such humins is based on synthetic model humins prepared at lab-scale from typical carbohydrate(-derived) compounds. Here, we report the first extensive characterisation study of an industrial humin. The soluble humin was generated from pilot plant-scale methanolic cyclodehydration of D-fructose to 5-methoxymethyl-2-furfural (MMF), as part of the Avantium YXY® process to produce FDCA. Purification of the industrial humin followed by fractionation allowed isolation of a water-insoluble, high molecular weight fraction (WIPIH) and a water-soluble, low-to-middle molecular weight soluble fraction (WES). Characterisation by elemental analysis, thermogravimetry, IR and NMR spectroscopy and size exclusion chromatography provided a detailed picture of the humin structure in both fractions. Aided by a comprehensive NMR spectral library of furanic model compounds, we identified the main furanic building blocks and inter-unit linkages and propose a structure for this industrial humin sample. The WIPIH and WES fractions were found to be composed of furanic rings interconnected by short aliphatic chains containing a wide range of functionalities including alcohols, ethers, carboxylic acids, esters, aldehydes and ketones. The low level of crosslinking and high functional group content of the industrial humin differ from the more extensively studied, (highly over-)condensed synthetic model humins, towards which they can be considered intermediates. The structural and compositional insights into the nature of an actual industrial humin open up a broad spectrum of valorisation opportunities., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Microtubules (MTs) are key components of the eukaryotic cytoskeleton and are essential for intracellular organization, organelle trafficking and mitosis. MT tasks depend on binding and interactions with MT-associated proteins (MAPs). MT-associated protein 7 (MAP7) has the unusual ability of both MT binding and activating kinesin-1-mediated cargo transport along MTs. Additionally, the protein is reported to stabilize MTs with its 112 amino-acid long MT-binding domain (MTBD). Here we investigate the structural basis of the interaction of MAP7 MTBD with the MT lattice. Using a combination of solid and solution-state nuclear magnetic resonance (NMR) spectroscopy with electron microscopy, fluorescence anisotropy and isothermal titration calorimetry, we shed light on the binding mode of MAP7 to MTs at an atomic level. Our results show that a combination of interactions between MAP7 and MT lattice extending beyond a single tubulin dimer and including tubulin C-terminal tails contribute to formation of the MAP7-MT complex., (© 2024. The Author(s).)

While plastics-to-plastics recycling via melting and re-extrusion is often the preferred option due to a relatively low CO 2 footprint, this technique requires a highly sorted waste stream and plastic properties can often not be maintained. Obtaining aromatics, such as benzene, toluene, and xylene (BTX), via catalytic pyrolysis of polyolefins, such as polypropylene and polyethylene, offers another attractive recycling technology. In this process, a discarded crude oil refinery catalyst (ECAT) was previously shown to lower the unwanted formation of deactivating coke species compared to a fresh crude oil refinery catalyst (FCC-cat), while yielding 20 wt% aromatics from polypropylene. In this work, we study the underlying reaction mechanism for this chemical recycling process over the fresh and used refinery catalyst as well as a model system, not containing any zeolite material, using a combination of microscopy and spectroscopy. More specifically, by using in situ fluorescence microscopy, in situ infrared spectroscopy, in situ ultraviolet-visible spectroscopy as well as ex situ solid-state nuclear magnetic resonance, we observe highly fluorescent methylated aromatic intermediates that differ for the three catalyst materials under study both in their fluorescence, IR, UV-vis, and NMR spectroscopy features. This detailed micro-spectroscopic comparison informs which potential reaction intermediates lead to increased coke formation. Our results suggests that a next generation of catalyst materials for this process would profit from a higher accessibility and a milder acidity compared to an FCC-cat and shows the great potential of using ECAT to reduce coking and obtain a BTX stream, which could be become the chemical building blocks for the manufacturing of e.g. , plastics and coating materials., Competing Interests: The authors declare no conflict of interest., (This journal is © The Royal Society of Chemistry.)

Antimicrobial resistance is a leading mortality factor worldwide. Here, we report the discovery of clovibactin, an antibiotic isolated from uncultured soil bacteria. Clovibactin efficiently kills drug-resistant Gram-positive bacterial pathogens without detectable resistance. Using biochemical assays, solid-state nuclear magnetic resonance, and atomic force microscopy, we dissect its mode of action. Clovibactin blocks cell wall synthesis by targeting pyrophosphate of multiple essential peptidoglycan precursors (C 55 PP, lipid II, and lipid III WTA ). Clovibactin uses an unusual hydrophobic interface to tightly wrap around pyrophosphate but bypasses the variable structural elements of precursors, accounting for the lack of resistance. Selective and efficient target binding is achieved by the sequestration of precursors into supramolecular fibrils that only form on bacterial membranes that contain lipid-anchored pyrophosphate groups. This potent antibiotic holds the promise of enabling the design of improved therapeutics that kill bacterial pathogens without resistance development., Competing Interests: Declaration of interests The following authors, A.J.P., C.A., A.N., A.L.S., L.L.L., D.H., and K.L., declare competing financial interests because they are employees and consultants of NovoBiotic Pharmaceuticals. A patent US 11,203,616 B2 was issued on 12/21/2021 and describes the use of clovibactin (Novo29) and as an antibiotic, as well as the pharmaceutical composition and antibiotic use of derivatives., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Studying the structural aspects of proteins within sub-cellular compartments is of growing interest. Dynamic nuclear polarization supported solid-state NMR (DNP-ssNMR) is uniquely suited to provide such information, but critically lacks the desired sensitivity and resolution. Here we utilize SNAPol-1, a novel biradical, to conduct DNP-ssNMR at high-magnetic fields (800 MHz/527 GHz) inside HeLa cells and isolated cell nuclei electroporated with [ 13 C, 15 N] labeled ubiquitin. We report that SNAPol-1 passively diffuses and homogenously distributes within whole cells and cell nuclei providing ubiquitin spectra of high sensitivity and remarkably improved spectral resolution. For cell nuclei, physical enrichment facilitates a further 4-fold decrease in measurement time and provides an exclusive structural view of the nuclear ubiquitin pool. Taken together, these advancements enable atomic interrogation of protein conformational plasticity at atomic resolution and with sub-cellular specificity., Competing Interests: The authors have no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

The cell wall fulfils several functions in the biology of fungi. For instance, it provides mechanical strength, interacts with the (a)biotic environment, and acts as a molecular sieve. Recently, it was shown that proteins and β-glucans in the cell wall of Schizophyllum commune bind Cu 2+ . We here show that the cell wall of this mushroom forming fungus also binds other (micro-)nutrients. Ca 2+ , Mg 2+ , Mn 2+ , NO 3 - , PO 4 3- , and SO 4 2- bound at levels > 1 mg per gram dry weight cell wall, while binding of BO 3 - , Cu 2+ , Zn 2+ and MoO 4 2- was lower. Sorption of Ca 2+ , Mn 2+ , Zn 2+ and PO 4 3- was promoted at alkaline pH. These compounds as well as BO 3 3- , Cu 2+ , Mg 2+ , NO 3 - , and SO 4 2- that had bound at pH 4, 6, or 8 could be released from the cell wall at pH 4 with a maximum efficiency of 46-93 %. Solid-state NMR spectroscopy showed that the metals had the same binding sites as Cu 2+ when a low concentration of this ion is used. Moreover, data indicate that anions bind to the cell wall as well as to the metal ions. Together, it is shown that the cell wall of S. commune binds various (micro-)nutrients and that this binding is higher than the uptake by hyphae. The binding to the cell wall may be used as a storage mechanism or may reduce availability of these molecules to competitors or prevent toxic influx in the cytoplasm., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Author(s).)

New technologies create new complexities. Since non-invasive prenatal tests (NIPTs) were first introduced, keeping pace with complexity constitutes an ongoing task for medical societies, politics, and practice. NIPTs analyse the chromosomes of the fetus from a small blood sample. Initially, NIPTs were targeted at detecting trisomy 21 (Down syndrome): meanwhile there are sequencing techniques capable of analysing the entire genome of the unborn child. These yield findings of unclear relevance for the child's future life, resulting in new responsibility structures and dilemmas for the parents-to-be.The industry's marketing strategies overemphasize the benefits of the tests while disregarding their consequences. This paper chooses the opposite path: starting with the underestimated consequences, it focuses on adverse developments and downsides. Disparities, paradoxes, and risks associated with NIPTs are illustrated, ethical conflicts described. Indications that new technologies developed to solve problems create new ones are examined. In the sense of critical thinking, seemingly robust knowledge is scrutinized for uncertainties and ambiguities. It analyses how the interplay between genetic knowledge and social discourse results in new dimensions of responsibility not only for parents-to-be, but also for decision-makers, authorities, and professional societies, illustrated by a review of different national policies and implementation programmes. As shown by the new NIPT policy in Norway, the consequences can be startling. Finally, a lawsuit in the United States illustrates how an agency can risk forfeiting its legitimation in connection with the inaccuracy of NIPTs., (© 2023. The Author(s).)

The microtubule-associated protein 7 (MAP7) is a protein involved in cargo transport along microtubules (MTs) by interacting with kinesin-1 through the C-terminal kinesin-binding domain. Moreover, the protein is reported to stabilize MT, thereby playing a key role in axonal branch development. An important element for this latter function is the 112 amino-acid long N-terminal microtubule-binding domain (MTBD) of MAP7. Here we report NMR backbone and side-chain assignments that suggest a primarily alpha-helical secondary fold of this MTBD in solution. The MTBD contains a central long α-helical segment that includes a short four-residue 'hinge' sequence with decreased helicity and increased flexibility. Our data represent a first step towards analysing the complex interaction of MAP7 with MTs at an atomic level via NMR spectroscopy., (© 2023. The Author(s).)

In the last three decades, the scope of solid-state NMR has expanded to exploring complex biomolecules, from large protein assemblies to intact cells at atomic-level resolution. This diversity in macromolecules frequently features highly flexible components whose insoluble environment precludes the use of solution NMR to study their structure and interactions. While High-resolution Magic-Angle Spinning (HR-MAS) probes offer the capacity for gradient-based 1 H-detected spectroscopy in solids, such probes are not commonly used for routine MAS NMR experiments. As a result, most exploration of the flexible regime entails either 13 C-detected experiments, the use of partially perdeuterated systems, or ultra-fast MAS. Here we explore proton-detected pulse schemes probing through-bond 13 C- 13 C networks to study mobile protein sidechains as well as polysaccharides in a broadband manner. We demonstrate the use of such schemes to study a mixture of microtubule-associated protein (MAP) tau and human microtubules (MTs), and the cell wall of the fungus Schizophyllum commune using 2D and 3D spectroscopy, to show its viability for obtaining unambiguous correlations using standard fast-spinning MAS probes at high and ultra-high magnetic fields., (© 2023. The Author(s).)

High strength, hardness, and fracture toughness are mechanical properties that are not commonly associated with the fleshy body of a fungus. Here, we show with detailed structural, chemical, and mechanical characterization that Fomes fomentarius is an exception, and its architectural design is a source of inspiration for an emerging class of ultralightweight high-performance materials. Our findings reveal that F . fomentarius is a functionally graded material with three distinct layers that undergo multiscale hierarchical self-assembly. Mycelium is the primary component in all layers. However, in each layer, mycelium exhibits a very distinct microstructure with unique preferential orientation, aspect ratio, density, and branch length. We also show that an extracellular matrix acts as a reinforcing adhesive that differs in each layer in terms of quantity, polymeric content, and interconnectivity. These findings demonstrate how the synergistic interplay of the aforementioned features results in distinct mechanical properties for each layer.

Solid-state NMR (ssNMR) spectroscopy facilitates the non-destructive characterization of structurally heterogeneous biomolecules in their native setting, for example, comprising proteins, lipids and polysaccharides. Here we demonstrate the utility of high and ultra-high field 1 H-detected fast MAS ssNMR spectroscopy, which exhibits increased sensitivity and spectral resolution, to further elucidate the atomic-level composition and structural arrangement of the cell wall of Schizophyllum commune, a mushroom-forming fungus from the Basidiomycota phylum. These advancements allowed us to reveal that Cu(II) ions and the antifungal peptide Cathelicidin-2 mainly bind to cell wall proteins at low concentrations while glucans are targeted at high metal ion concentrations. In addition, our data suggest the presence of polysaccharides containing N-acetyl galactosamine (GalNAc) and proteins, including the hydrophobin proteins SC3, shedding more light on the molecular make-up of cells wall as well as the positioning of the polypeptide layer. Obtaining such information may be of critical relevance for future research into fungi in material science and biomedical contexts., (© 2022 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

Cellular dynamic nuclear polarization (DNP) has been an effective means of overcoming the intrinsic sensitivity limitations of solid-state nuclear magnetic resonance (ssNMR) spectroscopy, thus enabling atomic-level biomolecular characterization in native environments. Achieving DNP signal enhancement relies on doping biological preparations with biradical polarizing agents (PAs). Unfortunately, PA performance within cells is often limited by their sensitivity to the reductive nature of the cellular lumen. Herein, we report the synthesis and characterization of a highly bioresistant and hydrophilic PA (StaPol-1) comprising the trityl radical OX063 ligated to a gem -diethyl pyrroline nitroxide via a rigid piperazine linker. EPR experiments in the presence of reducing agents such as ascorbate and in HeLa cell lysates demonstrate the reduction resistance of StaPol-1. High DNP enhancements seen in small molecules, proteins and cell lysates at 18.8 T confirm that StaPol-1 is an excellent PA for DNP ssNMR investigations of biomolecular systems at high magnetic fields in reductive environments., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

For almost five decades, solid-state NMR (ssNMR) has been used to study complex biomolecular systems. This article gives a view on how ssNMR methods and applications have evolved during this time period in a broader structural biology context. It also discusses possible directions for additional developments and the future role of ssNMR in a life science context and beyond., Competing Interests: The author declares that he has no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Author.)

Membrane proteins are known to exert many essential biological functions by forming complexes in cell membranes. An example refers to the β-barrel assembly machinery (BAM), a 200 kDa pentameric complex containing BAM proteins A-E that catalyzes the essential process of protein insertion into the outer membrane of gram-negative bacteria. While progress has been made in capturing three-dimensional structural snapshots of the BAM complex, the role of the lipoprotein BamC in the complex assembly in functional lipid bilayers has remained unclear. We have devised a component-selective preparation scheme to directly study BamC as part of the entire BAM complex in lipid bilayers. Combination with proton-detected solid-state NMR methods allowed us to probe the structure, dynamics, and supramolecular topology of full-length BamC embedded in the entire complex in lipid bilayers. Our approach may help decipher how individual proteins contribute to the dynamic formation and functioning of membrane protein complexes in membranes., (© 2022 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

The xanthophyll cycle in the antenna of photosynthetic organisms under light stress is one of the most well-known processes in photosynthesis, but its role is not well understood. In the xanthophyll cycle, violaxanthin (Vio) is reversibly transformed to zeaxanthin (Zea) that occupies Vio binding sites of light-harvesting antenna proteins. Higher monomer/trimer ratios of the most abundant light-harvesting protein, the light-harvesting complex II (LHCII), usually occur in Zea accumulating membranes and have been observed in plants after prolonged illumination and during high-light acclimation. We present a combined NMR and coarse-grained simulation study on monomeric LHCII from the npq2 mutant that constitutively binds Zea in the Vio binding pocket. LHCII was isolated from 13 C-enriched npq2 Chlamydomonas reinhardtii (Cr) cells and reconstituted in thylakoid lipid membranes. NMR results reveal selective changes in the fold and dynamics of npq2 LHCII compared with the trimeric, wild-type and show that npq2 LHCII contains multiple mono- or digalactosyl diacylglycerol lipids (MGDG and DGDG) that are strongly protein bound. Coarse-grained simulations on npq2 LHCII embedded in a thylakoid lipid membrane agree with these observations. The simulations show that LHCII monomers have more extensive lipid contacts than LHCII trimers and that protein-lipid contacts are influenced by Zea. We propose that both monomerization and Zea binding could have a functional role in modulating membrane fluidity and influence the aggregation and conformational dynamics of LHCII with a likely impact on photoprotection ability., (Copyright © 2021 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Dynamic nuclear polarization (DNP) is a powerful method to enhance the sensitivity of solid-state magnetic nuclear resonance (ssNMR) spectroscopy. However, its biomolecular applications at high magnetic fields (preferably>14 T) have so far been limited by the intrinsically low efficiency of polarizing agents and sample preparation aspects. Herein, we report a new class of trityl-nitroxide biradicals, dubbed SNAPols that combine high DNP efficiency with greatly enhanced hydrophilicity. SNAPol-1, the best compound in the series, shows DNP enhancement factors at 18.8 T of more than 100 in small molecules and globular proteins and also exhibits strong DNP enhancements in membrane proteins and cellular preparations. By integrating optimal sensitivity and high resolution, we expect widespread applications of this new polarizing agent in high-field DNP/ssNMR spectroscopy, especially for complex biomolecules., (© 2021 Wiley-VCH GmbH.)

Herein, we investigate a novel set of polarizing agents-mixed-valence compounds-by theoretical and experimental methods and demonstrate their performance in high-field dynamic nuclear polarization (DNP) NMR experiments in the solid state. Mixed-valence compounds constitute a group of molecules in which molecular mobility persists even in solids. Consequently, such polarizing agents can be used to perform Overhauser-DNP experiments in the solid state, with favorable conditions for dynamic nuclear polarization formation at ultra-high magnetic fields., (© 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

We report the supramolecular assembly of artificial metalloenzymes (ArMs), based on the Lactococcal multidrug resistance regulator (LmrR) and an exogeneous copper(II)-phenanthroline complex, in the cytoplasm of E. coli cells. A combination of catalysis, cell-fractionation, and inhibitor experiments, supplemented with in-cell solid-state NMR spectroscopy, confirmed the in-cell assembly. The ArM-containing whole cells were active in the catalysis of the enantioselective Friedel-Crafts alkylation of indoles and the Diels-Alder reaction of azachalcone with cyclopentadiene. Directed evolution resulted in two different improved mutants for both reactions, LmrR_A92E_M8D and LmrR_A92E_V15A, respectively. The whole-cell ArM system required no engineering of the microbial host, the protein scaffold, or the cofactor to achieve ArM assembly and catalysis. We consider this a key step towards integrating abiological catalysis with biosynthesis to generate a hybrid metabolism., (© 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Membrane proteins are vital for cell function and thus represent important drug targets. Solid-state Nuclear Magnetic Resonance (ssNMR) spectroscopy offers a unique access to probe the structure and dynamics of such proteins in biological membranes of increasing complexity. Here, we present modern solid-state NMR spectroscopy as a tool to study structure and dynamics of proteins in natural lipid membranes and at atomic scale. Such spectroscopic studies profit from the use of high-sensitivity ssNMR methods, i.e., proton-( 1 H)-detected ssNMR and DNP (Dynamic Nuclear Polarization) supported ssNMR. Using bacterial outer membrane beta-barrel protein BamA and the ion channel KcsA, we present methods to prepare isotope-labeled membrane proteins and to derive structural and motional information by ssNMR.

Curcumin-loaded polymeric micelles composed of poly(ethylene glycol)- b -poly( N -2-benzoyloxypropyl methacrylamide) (mPEG- b -p(HPMA-Bz)) were prepared to solubilize and improve the pharmacokinetics of curcumin. Curcumin-loaded micelles were prepared by a nanoprecipitation method using mPEG 5kDa - b -p(HPMA-Bz) copolymers with varying molecular weight of the hydrophobic block (5.2, 10.0, and 17.1 kDa). At equal curcumin loading, micelles composed of mPEG 5kDa - b -p(HPMA-Bz) 17.1kDa showed better curcumin retention in both phosphate-buffered saline (PBS) and plasma at 37 °C than micelles based on block copolymers with smaller hydrophobic blocks. No change in micelle size was observed during 24 h incubation in plasma using asymmetrical flow field-flow fractionation (AF 4 ), attesting to particle stability. However, 22-49% of the curcumin loading was released from the micelles during 24 h from formulations with the highest to the lowest molecular weight p(HPMA-Bz), respectively, in plasma. AF 4 analysis further showed that the released curcumin was subsequently solubilized by albumin. In vitro analyses revealed that the curcumin-loaded mPEG 5kDa - b -p(HPMA-Bz) 17.1kDa micelles were internalized by different types of cancer cells, resulting in curcumin-induced cell death. Intravenously administered curcumin-loaded, Cy7-labeled mPEG 5kDa - b -p(HPMA-Bz) 17.1kDa micelles in mice at 50 mg curcumin/kg showed a long circulation half-life for the micelles ( t 1/2 = 42 h), in line with the AF 4 results. In contrast, the circulation time of curcumin was considerably shorter than that of the micelles ( t 1/2α = 0.11, t 1/2β = 2.5 h) but ∼5 times longer than has been reported for free curcumin ( t 1/2α = 0.02 h). The faster clearance of curcumin in vivo compared to in vitro studies can be attributed to the interaction of curcumin with blood cells. Despite the excellent solubilizing effect of these micelles, no cytostatic effect was achieved in neuroblastoma-bearing mice, possibly because of the low sensitivity of the Neuro2A cells to curcumin.