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Regulation of DNA-templated processes such as gene transcription and DNA repair depend on the interaction of a wide range of proteins with the nucleosome, the fundamental building block of chromatin. Both solution and solid-state NMR spectroscopy have become an attractive approach to study the dynamics and interactions of nucleosomes, despite their high molecular weight of ∼200 kDa. For solid-state NMR (ssNMR) studies, dilute solutions of nucleosomes are converted to a dense phase by sedimentation or precipitation. Since nucleosomes are known to self-associate, these dense phases may induce extensive interactions between nucleosomes, which could interfere with protein-binding studies. Here, we characterized the packing of nucleosomes in the dense phase created by sedimentation using NMR and small-angle X-ray scattering (SAXS) experiments. We found that nucleosome sediments are gels with variable degrees of solidity, have nucleosome concentration close to that found in crystals, and are stable for weeks under high-speed magic angle spinning (MAS). Furthermore, SAXS data recorded on recovered sediments indicate that there is no pronounced long-range ordering of nucleosomes in the sediment. Finally, we show that the sedimentation approach can also be used to study low-affinity protein interactions with the nucleosome. Together, our results give new insights into the sample characteristics of nucleosome sediments for ssNMR studies and illustrate the broad applicability of sedimentation-based NMR studies.

Matrix effects in a fluid catalytic cracking (FCC) catalyst have been studied in terms of structure, accessibility, and acidity. An extensive characterization study into the structural and acidic properties of a FCC catalyst, its individual components (i.e., zeolite H‐Y, binder (boehmite/silica) and kaolin clay), and two model FCC catalyst samples containing only two components (i.e., zeolite‐binder and binder‐clay) was performed at relevant conditions. This allowed the drawing of conclusions about the role of each individual component, describing their mutual physicochemical interactions, establishing structure‐acidity relationships, and determining matrix effects in FCC catalyst materials. This has been made possible by using a wide variety of characterization techniques, including temperature‐programmed desorption of ammonia, infrared spectroscopy in combination with CO as probe molecule, transmission electron microscopy, X‐ray diffraction, Ar physisorption, and advanced nuclear magnetic resonance. By doing so it was, for example, revealed that a freshly prepared spray‐dried FCC catalyst appears as a physical mixture of its individual components, but under typical riser reactor conditions, the interaction between zeolite H‐Y and binder material is significant and mobile aluminum migrates and inserts from the binder into the defects of the zeolite framework, thereby creating additional Brønsted acid sites and restoring the framework structure., On the interplay between matrix and catalyst: A combination of advanced characterization techniques reveals that while a freshly prepared spray‐dried fluid catalytic cracking catalyst appears as a physical mixture of its individual components, significant matrix effects actually come into play under industrial riser reactor conditions, leading to changing Brønsted and Lewis acidity, pore accessibility as well as various structural changes.

Microtubules are important components of the eukaryotic cytoskeleton. Their structural organization is regulated by nucleotide binding and many microtubule-associated proteins (MAPs). While cryo-EM and X-ray crystallography have provided detailed views of interactions between MAPs with the microtubule lattice, little is known about how MAPs and their intrinsically disordered regions interact with the dynamic microtubule surface. NMR carries the potential to directly probe such interactions but so far has been precluded by the low tubulin yield. We present a protocol to produce [13C, 15N]-labeled, functional microtubules (MTs) from human cells for solid-state NMR studies. This approach allowed us to demonstrate that MAPs can differently modulate the fast time-scale dynamics of C-terminal tubulin tails, suggesting distinct interaction modes. Our results pave the way for in-depth NMR studies of protein dynamics involved in MT assembly and their interactions with other cellular components., Microtubule (MT) organization is regulated by many microtubule-associated proteins (MAPs) that contain intrinsically disordered regions. Here authors produce [13C, 15N] labeled, functional microtubules from human cells for solid-state NMR which allows studying MAP-MT interactions.

The modes of deactivation -and the extent to which their properties can be restored- of two catalyst bodies used in cascade for bio-oil synthesis have been studied. These catalysts include a solid acid granulate (namely ZrO2/desilicated zeolite ZSM-5/attapulgite clay) employed in ex-situ catalytic fast pyrolysis of biomass, and a base extrudate (K-exchanged zeolite USY/attapulgite clay) for the subsequent bio-oil upgrading. Post-mortem analyses of both catalyst bodies with Raman spectroscopy and confocal fluorescence microscopy revealed the presence of highly poly-aromatic coke distributed in an egg-shell manner. Deactivation due to coke adsorption onto acid sites affected the zeolite ZSM-5-based catalyst, while for the base catalyst it is structural integrity loss, resulting from KOH-mediated zeolite framework collapse, the main deactivating factor. A hydrothermal regeneration process reversed the detrimental effects of coke in the acid catalyst, largely recovering catalyst acidity (∼80%) and textural properties (∼90%), but worsened the structural damage suffered by the base catalyst.

The nucleosome surface contains an area with negative electrostatic potential known as the acidic patch, which functions as a binding platform for various proteins to regulate chromatin biology. The dense clustering of acidic residues may impact their effective pKa and thus the electronegativity of the acidic patch, which in turn could influence nucleosome-protein interactions. We here set out to determine the pKa values of residues in and around the acidic patch in the free H2A-H2B dimer using NMR spectroscopy. We present a refined solution structure of the H2A-H2B dimer based on intermolecular distance restraints, displaying a well-defined histone-fold core. We show that the conserved histidines H2B H46 and H106 that line the acidic patch have pKa of 5.9 and 6.5, respectively, and that most acidic patch carboxyl groups have pKa values well below 5.0. For H2A D89 we find strong evidence for an elevated pKa of 5.3. Our data establish that the acidic patch is highly negatively charged at physiological pH, while protonation of H2B H106 and H2B H46 at slightly acidic pH will reduce electronegativity. These results will be valuable to understand the impact of pH changes on nucleosome-protein interactions in vitro, in silico or in vivo.

Lipo-tridecapeptides, a class of bacterial non-ribosomally produced peptides, show strong antimicrobial activity against Gram-positive pathogens, including antibiotic-resistant Staphylococcus aureus and Enterococcus spp. However, many of these lipo-tridecapeptides have shown high hemolytic activity and cytotoxicity, which has limited their potential to be developed into antibiotics. Recently, we reported a novel antimicrobial lipo-tridecapeptide, brevibacillin 2V, which showed no hemolytic activity against human red blood cells at a high concentration of 128 mg/L, opposite to other brevibacillins and lipo-tridecapeptides. In addition, brevibacillin 2V showed much lower cytotoxicity than the other members of the brevibacillin family. In this study, we set out to elucidate the antimicrobial mode of action of brevibacillin 2V. The results show that brevibacillin 2V acts as bactericidal antimicrobial agent against S. aureus (MRSA). Further studies show that brevibacillin 2V exerts its bactericidal activity by binding to the bacterial cell wall synthesis precursor Lipid II and permeabilizing the bacterial membrane. Combined solid-state NMR, circular dichroism, and isothermal titration calorimetry assays indicate that brevibacillin 2V binds to the GlcNAc-MurNAc moiety and/or the pentapeptide of Lipid II. This study provides an insight into the antimicrobial mode of action of brevibacillin 2V. As brevibacillin 2V is a novel and promising antibiotic candidate with low hemolytic activity and cytotoxicity, the here-elucidated mode of action will help further studies to develop it as an alternative antimicrobial agent.

Bacterial non-ribosomally produced peptides (NRPs) form a rich source of antibiotics, including more than 20 of these antibiotics that are used in the clinic, such as penicillin G, colistin, vancomycin, and chloramphenicol. Here we report the identification, purification, and characterization of a novel NRP, i.e., brevibacillin 2V (lipo-tridecapeptide), from Brevibacillus laterosporus DSM 25. Brevibacillin 2V has a strong antimicrobial activity against Gram-positive bacterial pathogens (minimum inhibitory concentration = 2 mg/L), including difficult-to-treat antibiotic-resistant Enterococcus faecium, Enterococcus faecalis, and Staphylococcus aureus. Notably, brevibacillin 2V has a much lower hemolytic activity (HC50 > 128 mg/L) and cytotoxicity (CC50 = 45.49 ± 0.24 mg/L) to eukaryotic cells than previously reported NRPs of the lipo-tridecapeptide family, including other brevibacillins, which makes it a promising candidate for antibiotic development. In addition, our results demonstrate that brevibacillins display a synergistic action with established antibiotics against Gram-negative bacterial pathogens. Probably due to the presence of non-canonical amino acids and D-amino acids, brevibacillin 2V showed good stability in human plasma. Thus, we identified and characterized a novel and promising antimicrobial candidate (brevibacillin 2V) with low hemolytic activity and cytotoxicity, which can be used either on its own or as a template for further total synthesis and modification.

De novo macrocyclic peptides, derived using selection technologies such as phage and mRNA display, present unique and unexpected solutions to challenging biological problems. This is due in part to their unusual folds, which are able to present side chains in ways not available to canonical structures such as α-helices and β-sheets. Despite much recent interest in these molecules, their folding and binding behavior remains poorly characterized. In this work, we present cocrystallization, docking, and solution NMR structures of three de novo macrocyclic peptides that all bind as competitive inhibitors with single-digit nanomolar K i to the active site of human pancreatic α-amylase. We show that a short stably folded motif in one of these is nucleated by internal hydrophobic interactions in an otherwise dynamic conformation in solution. Comparison of the solution structures with a target-bound structure from docking indicates that stabilization of the bound conformation is provided through interactions with the target protein after binding. These three structures also reveal a surprising functional convergence to present a motif of a single arginine sandwiched between two aromatic residues in the interactions of the peptide with the key catalytic residues of the enzyme, despite little to no other structural homology. Our results suggest that intramolecular hydrophobic interactions are important for priming binding of small macrocyclic peptides to their target and that high rigidity is not necessary for high affinity.

Spontaneous activity shifts at constant experimental conditions represent a widespread regulatory mechanism in ion channels. The molecular origins of these modal gating shifts are poorly understood. In the K+ channel KcsA, a multitude of fast activity shifts that emulate the native modal gating behaviour can be triggered by point-mutations in the hydrogen bonding network that controls the selectivity filter. Using solid-state NMR and molecular dynamics simulations in a variety of KcsA mutants, here we show that modal gating shifts in K+ channels are associated with important changes in the channel dynamics that strongly perturb the selectivity filter equilibrium conformation. Furthermore, our study reveals a drastically different motional and conformational selectivity filter landscape in a mutant that mimics voltage-gated K+ channels, which provides a foundation for an improved understanding of eukaryotic K+ channels. Altogether, our results provide a high-resolution perspective on some of the complex functional behaviour of K+ channels.

Photosynthetic light-harvesting complexes of higher plants, moss and green algae can undergo dynamic conformational transitions, which have been correlated to their ability to adapt to fluctuations in the light environment. Herein, we demonstrate the application of solid-state NMR spectroscopy on native, heterogeneous thylakoid membranes of Chlamydomonas reinhardtii (Cr) and on Cr Light-Harvesting Complex II (LHCII) in thylakoid lipid bilayers to detect LHCII conformational dynamics in its native membrane environment. We show that membrane-reconstituted LHCII contains selective sites that undergo fast, large-amplitude motions, including the phytol tails of two chlorophylls. Protein plasticity is also observed in the N-terminal stromal loop and in protein fragments facing the lumen, involving sites that stabilize the xanthophyll-cycle carotenoid violaxanthin and the two luteins. The results report on the intrinsic flexibility of LHCII pigment-protein complexes in a membrane environment, revealing putative sites for conformational switching. In thylakoid membranes, fast dynamics of protein and pigment sites is significantly reduced, which suggests that in their native organelle membranes, LHCII complexes are locked in specific conformational states.STATEMENT OF SIGNIFICANCEPhotosynthetic Light-Harvesting Complexes undergo dynamic conformational transitions that regulate the capacity of the light-harvesting antenna. We demonstrate the application of solid-state (ss)NMR spectroscopy to investigate the structural dynamics of LHCII, the most abundant LHC complex of plants and algae, in native membranes. Selective dynamic protein and pigment residues are identified that are putative sites for a conformational switch.

Catalyst deactivation due to coking is a major challenge in the catalytic fast pyrolysis (CFP) of biomass. Here, a multitechnique investigation of a technical Al2O3-bound ZSM-5-based extrudate catalyst, used for the CFP of pine wood and cellulose (at a reactor temperature of 500 °C), provided insight into the effects of extrusion, the catalytic pyrolysis process, and catalyst regeneration on the catalyst structure. As a result of a reduction in acidity and surface area due to the coking catalyst, the activity dropped drastically with increasing time-on-stream (TOS), as evidenced by a decrease in aromatics yield. Strikingly, confocal fluorescence microscopy at the single-particle level revealed that vapor components derived from whole biomass or just the cellulose component coke differently. While pine-wood-derived species mainly blocked the external area of the catalyst particle, larger carbon deposits were formed inside the catalyst's micropores with cellulose-derived species. Pyridine FT-IR and solid-state NMR spectroscopy demonstrated irreversible changes after regeneration, likely due to partial dealumination. Taken together with

Membrane proteins are among the most challenging systems to study with experimental structural biology techniques. The increased number of deposited structures of membrane proteins has opened the route to modeling their complexes by methods such as docking. Here, we present an integrative computational protocol for the modeling of membrane-associated protein assemblies. The information encoded by the membrane is represented by artificial beads, which allow targeting of the docking toward the binding-competent regions. It combines efficient, artificial intelligence-based rigid-body docking by LightDock with a flexible final refinement with HADDOCK to remove potential clashes at the interface. We demonstrate the performance of this protocol on eighteen membrane-associated complexes, whose interface lies between the membrane and either the cytosolic or periplasmic regions. In addition, we provide a comparison to another state-of-the-art docking software, ZDOCK. This protocol should shed light on the still dark fraction of the interactome consisting of membrane proteins., Most approaches for modeling the membrane protein complexes are not capable of incorporating the topological information provided by the membrane. Here authors present an integrative computational protocol for the modeling of membrane-associated protein assemblies, specifically complexes consisting of a membrane-embedded protein and a soluble partner.

Cell walls are essential in the interaction of fungi with the (a)biotic environment and are also key to hyphal morphogenesis and mechanical strength. Here, we used solid-state NMR (ssNMR) spectroscopy combined with HPLC and GC–MS to study the structural organization of the cell wall of a representative of the Basidiomycota, one of the two main phyla of fungi. Based on the data we propose a refined model for the cell wall of a basidiomycete. In this model, the rigid core is built from α- and β-(1,3)-glucan, β-(1,3)-(1,6)-glucan, highly branched and single stranded β-(1,4)-chitin as well as polymeric fucose. The mobile fraction of the cell wall is composed of β-(1,3)-glucan, β-(1,3)-(1,6)-glucan, β-(1,6)-glucan, α-linked reducing and non-reducing ends and polymeric mannose. Together, these findings provide novel insights into the structural organization of the cell wall of the model basidiomycete S. commune that was previously based on destructive chemical and enzymatic analysis.

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., Artificial metalloenzymes were created by supramolecular assembly in the cytoplasm of E. coli cells. The cells were then applied to the enantioselective biocatalysis of a Friedel–Crafts alkylation of indoles (see picture) and a Diels–Alder reaction. Directed evolution of the artificial metalloenzymes inside the cells provided improved variants for both whole‐cell biocatalytic reactions.

Crosslinking mass spectrometry (Crosslinking MS) has substantially matured as a method over the last two decades through parallel development in multiple labs, demonstrating its applicability for protein structure determination, conformation analysis and mapping protein interactions in complex mixtures. Crosslinking MS has become a much-appreciated and routinely applied tool especially in structural biology. Therefore, it is timely that the community commits to the development of methodological and reporting standards. This white paper builds on an open process comprising a number of events at community conferences since 2015 and identifies aspects of Crosslinking MS for which guidelines should be developed as part of a Crosslinking MS standards initiative., Comment: 26 pages, 1 figure

Targeting of proteins in the histone modification machinery has emerged as a promising new direction to fight disease. The search for compounds that inhibit proteins that readout histone modification has led to several new epigenetic drugs, mostly for proteins involved in recognition of acetylated lysines. However, this approach proved to be a challenging task for methyllysine readers, which typically feature shallow binding pockets. Moreover, reader proteins of trimethyllysine K36 on the histone H3 (H3K36me3) not only bind the methyllysine but also the nucleosomal DNA. Here, we sought to find peptide-based binders of H3K36me3 reader PSIP1, which relies on DNA interactions to tightly bind H3K36me3 modified nucleosomes. We designed several peptides that mimic the nucleosomal context of H3K36me3 recognition by including negatively charged Glu-rich regions. Using a detailed NMR analysis, we find that addition of negative charges boosts binding affinity up to 50-fold while decreasing binding to the trimethyllysine binding pocket. Since screening and selection of compounds for reader domains is typically based solely on affinity measurements due to their lack of enzymatic activity, our case highlights the need to carefully control for the binding mode, in particular for the challenging case of H3K36me3 readers.

Dynamic nuclear polarization (DNP) is a powerful method to enhance nuclear magnetic resonance (NMR) signal intensities, enabling unprecedented applications in life and material science. An ultimate goal is to expand the use of DNP-enhanced solid-state NMR to ultrahigh magnetic fields where optimal spectral resolution and sensitivity are integrated. Trityl-nitroxide (TN) biradicals have attracted significant interest in high-field DNP, but their application to complex (bio)molecules has so far been limited. Here we report a novel postmodification strategy for synthesis of hydrophilic TN biradicals in order to improve their use in biomolecular applications. Initially, three TN biradicals (referred to as NATriPols 1-3) with amino-acid linkers were synthesized. EPR studies showed that the α-position of the amino-acid linkers is an ideal modification site for these biradicals since their electron-electron magnetic interactions are marginally affected by the substituents at this position. On the basis of this finding, we synthesized NATriPol-4 with pyridine disulfide appended at the α-position. Postmodification of NATriPol-4 via thiol-click chemistry resulted in various TN biradicals including hydrophilic NATriPol-5 in a quantitative manner. Interestingly, DNP enhancements at 18.8 T of NATriPols for 13C,15N-proline in a glycerol/water matrix are inversely correlated with their hydrophobicity. Importantly, applications of hydrophilic NATriPol-5 and NATriPol-3 to biomolecules including a globular soluble protein and a membrane targeting peptide reveal significantly improved performance compared to TEMTriPol-1 and AMUPol. Our work provides an efficient approach for one-step synthesis of new polarizing agents with tunable physicochemical properties, thus expediting optimization of new biradicals for biomolecular applications at ultrahigh magnetic fields.

We report on the unexpected finding that click modification of iduronyl azides results in a conformational flip of the pyranose ring, which led to the development of a new strategy for the design of superior enzyme substrates for the diagnostic assaying of iduronate-2-sulfatase (I2S), a lysosomal enzyme related to Hunter syndrome. Synthetic substrates are essential in testing newborns for metabolic disorders to enable early initiation of therapy. Our click-flipped iduronyl triazole showed a remarkably better performance with I2S than commonly used O-iduronates. We found that both O- and triazole-linked substrates are accepted by the enzyme, irrespective of their different conformations, but only the O-linked product inhibits the activity of I2S. Thus, in the long reaction times required for clinical assays, the triazole substrate substantially outperforms the O-iduronate. Applying our click-flipped substrate to assay I2S in dried blood spots sampled from affected patients and random newborns significantly increased the confidence in discriminating between these groups, clearly indicating the potential of the click-flip strategy to control the biomolecular function of carbohydrates., Click-triggered flip of the conformation of a sulfated iduronyl azide afforded a superior enzyme substrate to screen for Hunter syndrome.

Recent advances in the field of in-cell NMR spectroscopy have made it possible to study proteins in the context of bacterial or mammalian cell extracts or even entire cells. As most mammalian cells are part of a multi-cellular complex, there is a need to develop novel NMR approaches enabling the study of proteins within the complexity of a 3D cellular environment. Here we investigate the use of the hanging drop method to grow spheroids which are homogenous in size and shape as a model system to study solid tumors using solid-state NMR (ssNMR) spectroscopy. We find that these spheroids are stable under magic-angle-spinning conditions and show a clear change in metabolic profile as compared to single cell preparations. Finally, we utilize dynamic nuclear polarization (DNP)-supported ssNMR measurements to show that low concentrations of labelled nanobodies targeting EGFR (7D12) can be detected inside the spheroids. These findings suggest that solid-state NMR can be used to directly examine proteins or other biomolecules in a 3D cellular microenvironment with potential applications in pharmacological research.

We report the performance of our newly introduced Ensemble Docking with Enhanced sampling of pocket Shape (EDES) protocol coupled to a template-based algorithm to generate near-native ligand conformations in the 2019 iteration of the Grand Challenge organized by the D3R consortium. Using either AutoDock4.2 or HADDOCK2.2 docking programs (each software in two variants of the protocol) our method generated native-like poses among the top 5 submitted for evaluation for most of the 20 targets with similar performances. The protein selected for GC4 was the human beta-site amyloid precursor protein cleaving enzyme 1 (BACE-1), a transmembrane aspartic-acid protease. We identified at least one pose whose heavy-atoms RMSD was less than 2.5 {\AA} from the native conformation for 16 (80%) and 17 (85%) of the twenty targets using AutoDock and HADDOCK, respectively. Dissecting the possible sources of errors revealed that: i) our EDES protocol (with minor modifications) was able to sample sub-{\aa}ngstrom conformations for all 20 protein targets, reproducing the correct conformation of the binding site within ~1 {\AA} RMSD; ii) as already shown by some of us in GC3, even in the presence of near-native protein structures, a proper selection of ligand conformers is crucial for the success of ensemble-docking calculations. Importantly, our approach performed best among the protocols exploiting only structural information of the apo protein to generate conformations of the receptor for ensemble-docking calculations.

The authors gratefully acknowledge financial support of NWO, the Smart Mix Program of the Netherlands Ministry of Economic Affairs and the Netherlands Ministry of Education, Culture and Science. The NWO Large grant 175.107.301.10 is also gratefully acknowledged. Kraft lignin, the main by-product of the pulping industry, is an abundant, yet highly underutilized renewable aromatic polymer. During kraft pulping, the lignin undergoes extensive structural modification, with many labile native bonds being replaced by new, more recalcitrant ones. Currently little is known about the nature of those bonds and linkages in kraft lignin, information that is essential for its efficient valorization to renewable fuels, materials or chemicals. Here, we provide detailed new insights into the structure of softwood kraft lignin, identifying and quantifying the major native as well as kraft pulping-derived units as a function of molecular weight. De novo synthetic kraft lignins, generated from (isotope labelled) dimeric and advanced polymeric models, provided key mechanistic understanding of kraft lignin formation, revealing different process dependent reaction pathways to be operating. The discovery of a novel kraft-derived lactone condensation product proved diagnostic for the identification of a previously unknown homovanillin based condensation pathway. The lactone marker is found in various different soft- and hardwood kraft lignins, suggesting the general pertinence of this new condensation mechanism for kraft pulping. These novel structural and mechanistic insights will aid the development of future biomass and lignin valorization technologies. Publisher PDF

Methane dehydroaromatization (MDA) is among the most challenging processes in catalysis science owing to the inherent harsh reaction conditions and fast catalyst deactivation. To improve this process, understanding the mechanism of the initial C−C bond formation is essential. However, consensus about the actual reaction mechanism is still to be achieved. In this work, using advanced magic-angle spinning (MAS) solid-state NMR spectroscopy, we study in detail the early stages of the reaction over a well-dispersed Mo/H-ZSM-5 catalyst. Simultaneous detection of acetylene (i.e., presumably the direct C−C bond-forming product from methane), methylidene, allenes, acetal, and surface-formate species, along with the typical olefinic/aromatic species, allow us to conclude the existence of at least two independent C−H activation pathways. Moreover, this study emphasizes the significance of mobility-dependent host–guest chemistry between an inorganic zeolite and its trapped organic species during heterogeneous catalysis.

The natural antibiotic teixobactin kills pathogenic bacteria without detectable resistance. The difficult synthesis and unfavourable solubility of teixobactin require modifications, yet insufficient knowledge on its binding mode impedes the hunt for superior analogues. Thus far, teixobactins are assumed to kill bacteria by binding to cognate cell wall precursors (Lipid II and III). Here we present the binding mode of teixobactins in cellular membranes using solid-state NMR, microscopy, and affinity assays. We solve the structure of the complex formed by an improved teixobactin-analogue and Lipid II and reveal how teixobactins recognize a broad spectrum of targets. Unexpectedly, we find that teixobactins only weakly bind to Lipid II in cellular membranes, implying the direct interaction with cell wall precursors is not the sole killing mechanism. Our data suggest an additional mechanism affords the excellent activity of teixobactins, which can block the cell wall biosynthesis by capturing precursors in massive clusters on membranes., The natural antibiotic teixobactin kills bacteria by direct binding to their cognate cell wall precursors (Lipid II and III). Here authors use solid-state NMR to reveal the native binding mode of teixobactins and show that teixobactins only weakly bind to Lipid II in anionic cellular membranes.

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.

Background Antimicrobial peptides are considered potential alternatives for antibiotics. Here we describe the antibacterial properties of a family of novel cathelicidin-related (CR-) peptides, which we named PepBiotics, against bacteria typically present in cystic fibrosis (CF) patients. Methods Broth dilution assays were used to determine antibacterial activity of PepBiotics under physiological conditions, as well as development of bacterial resistance against these peptides. Toxicity was tested in mice and cell cultures while molecular interactions of Pepbiotics with bacterial membrane components was determined using CD, ITC and LPS/LTA induced macrophage studies. Results A relatively small number of PepBiotics remained highly antibacterial against CF-related respiratory pathogens Pseudomonas aeruginosa and Staphylococcus aureus, at high ionic strength and low pH. Interestingly, these PepBiotics also prevented LPS/LTA induced activation of macrophages and was shown to be non-toxic to primary human nasal epithelial cells. Furthermore, both P. aeruginosa and S. aureus were unable to induce resistance against CR-163 and CR-172, two PepBiotics selected for their excellent antimicrobial and immunomodulatory properties. Toxicity studies in mice indicated that intratracheal administration of CR-163 was well tolerated in vivo. Finally, interaction of CR-163 with bacterial-type anionic membranes but not with mammalian-type (zwitterionic lipid) membranes was confirmed using isothermal titration calorimetry and 31P solid state NMR. Conclusions PepBiotics are a promising novel class of highly active antimicrobial peptides, of which CR-163 showed the most potential for treatment of clinically relevant (CF-) pathogens in physiological conditions. General significance These observations emphasize the therapeutic potential of PepBiotics against CF-related bacterial respiratory infections.

The synthesis of many industrial bulk and fine chemicals frequently involves electrophilic aromatic substitution (SEAr) reactions. The most widely practiced example of the SEAr mechanism is the zeolite-catalysed ethylation of benzene, using ethylene as an alkylating agent. However, the current production route towards ethylbenzene is completely dependent on fossil resources, making the recent commercial successes in the zeolite-catalysed benzene ethylation process using bioethanol (instead of ethylene) very encouraging and noteworthy. Unfortunately, there is no information available on the reaction mechanism of this alternative synthesis route. Here, by employing a combination of advanced solid-state NMR spectroscopy and operando UV-Vis diffuse reflectance spectroscopy with on-line mass spectrometry, we have obtained detailed mechanistic insights into the bioethanol-mediated benzene ethylation process through the identification of active surface ethoxy species, surface-adsorbed zeolite–aromatic π-complexes, as well as the more controversial Wheland-type σ-complex. Moreover, we distinguish between rigid and mobile zeolite-trapped organic species, providing further evidence for distinctive host–guest chemistry during catalysis.

Liquid–liquid phase separation is increasingly recognized as a process involved in cellular organization. Thus far, a detailed structural characterization of this intrinsically heterogeneous process has been challenging. Here we combine solid- and solution-state NMR spectroscopy to obtain atomic-level insights into the assembly and maturation of cytoplasmic processing bodies that contain mRNA as well as enzymes involved in mRNA degradation. In detail, we have studied the enhancer of decapping 3 (Edc3) protein that is a central hub for processing body formation in yeast. Our results reveal that Edc3 domains exhibit diverse levels of structural organization and dynamics after liquid–liquid phase separation. In addition, we find that interactions between the different Edc3 domains and between Edc3 and RNA in solution are largely preserved in the condensed protein state, allowing processing bodies to rapidly form and dissociate upon small alterations in the cellular environment., Processing bodies are membrane less organelles that contain enzymes involved in mRNA turnover, among them enhancer of decapping 3 (Edc3). Here the authors use solid- and solution-state NMR spectroscopy to characterize the structural organization and dynamics of Edc3 and find that its interactions with RNA and between the different Edc3 domains are largely preserved in the phase-separated state.

Elucidating at atomic level how proteins interact and are chemically modified in cells represents a leading frontier in structural biology. We have developed a tailored solid-state NMR spectroscopic approach that allows studying protein structure inside human cells at atomic level under high-sensitivity dynamic nuclear polarization (DNP) conditions. We demonstrate the method using ubiquitin (Ub), which is critically involved in cellular functioning. Our results pave the way for structural studies of larger proteins or protein complexes inside human cells, which have remained elusive to in-cell solution-state NMR spectroscopy due to molecular size limitations.

Predicting the 3D structure of protein interactions remains a challenge in the field of computational structural biology. This is in part due to difficulties in sampling the complex energy landscape of multiple interacting flexible polypeptide chains. Coarse-graining approaches, which reduce the number of degrees of freedom of the system, help address this limitation by smoothing the energy landscape, allowing an easier identification of the global energy minimum. They also accelerate the calculations, allowing to model larger assemblies. Here, we present the implementation of the MARTINI coarse-grained force field for proteins into HADDOCK, our integrative modelling platform. Docking and refinement are performed at the coarse-grained level and the resulting models are then converted back to atomistic resolution through a distance restraints-guided morphing procedure. Our protocol, tested on the largest complexes of the protein docking benchmark 5, shows an overall ~7-fold speed increase compared to standard all-atom calculations, while maintaining a similar accuracy and yielding substantially more near-native solutions. To showcase the potential of our method, we performed simultaneous 7 body docking to model the 1:6 KaiC-KaiB complex, integrating mutagenesis and hydrogen/deuterium exchange data from mass spectrometry with symmetry restraints, and validated the resulting models against a recently published cryo-EM structure.

International audience; Zeolite-based catalysts are globally employed in many industrial processes, such as in crude-oil refining and in the production of bulk chemicals. However, to be implemented in industrial reactors efficiently, zeolite powders are required to be shaped in catalyst bodies. Scale-up of zeolite catalysts into such forms comes with side effects to its overall physicochem-ical properties and to those of its constituting components. Although fundamental research into "technical" solid catalysts is scarce, binder effects have been reported to significantly impact their catalytic properties and lifetime. Given the large number of additional (in)organic components added in the formulation, it is somehow surprising to see that there is a distinct lack of research into the unintentional impact organic additives can have on the properties of the zeolite and the catalyst bodies in general. Here, we systematically prepared a series of alumina-bound zeolite ZSM-5-based catalyst bodies, with organic additives such as peptizing, plasticizing, and lubricating agents, to rationalize their impacts on the physicochemical properties of the shaped catalyst bodies. By utilizing a carefully selected arsenal of bulk and high-spatial resolution multiscale characterization techniques, as well as specifically sized bioinspired fluorescent nanoprobes to study pore accessibility, we clearly show that, although the organic additives achieve their primary function of a mechanically robust material, uncontrolled processes are taking place in parallel. We reveal that the extrusion process can lead to zeolite dealumination (from acid peptizing treatment, and localized steaming upon calcination); meso-and macropore structural rearrangement (via burning-out of organic plasticizing and lubricating agents upon calcination); and abating of known alumina binder effects (via scavenging of Al species via chelating lubricating agents), which significantly impact catalytic performance. Understanding the mechanisms behind such effects in industrial-grade catalyst formulations can lead to enhanced design of these important materials, which can improve process efficiency in a vast range of industrial catalytic reactions.

CAMSAP/Patronins regulate microtubule minus-end dynamics. Their end specificity is mediated by their CKK domains, which we proposed recognise specific tubulin conformations found at minus ends. To critically test this idea, we compared the human CAMSAP1 CKK domain (HsCKK) with a CKK domain from Naegleria gruberi (NgCKK), which lacks minus-end specificity. Here we report near-atomic cryo-electron microscopy structures of HsCKK- and NgCKK-microtubule complexes, which show that these CKK domains share the same protein fold, bind at the intradimer interprotofilament tubulin junction, but exhibit different footprints on microtubules. NMR experiments show that both HsCKK and NgCKK are remarkably rigid. However, whereas NgCKK binding does not alter the microtubule architecture, HsCKK remodels its microtubule interaction site and changes the underlying polymer structure because the tubulin lattice conformation is not optimal for its binding. Thus, in contrast to many MAPs, the HsCKK domain can differentiate subtly specific tubulin conformations to enable microtubule minus-end recognition., CKK domain containing CAMSAP/Patronins recognise and regulate microtubule (MT) minus end dynamics. Here the authors compare cryo-EM structures of MT-bound human CKK and Naegleria gruberi CKK which lacks minus-end binding preference, finding NgCKK has a different interaction with, and inability to remodel, its MT binding site, shedding light on the CAMSAP/Patronin end binding mechanism.

Although industrialized, the mechanism for catalytic upgrading of bioethanol over solid‐acid catalysts (that is, the ethanol‐to‐hydrocarbons (ETH) reaction) has not yet been fully resolved. Moreover, mechanistic understanding of the ETH reaction relies heavily on its well‐known “sister‐reaction” the methanol‐to‐hydrocarbons (MTH) process. However, the MTH process possesses a C1‐entity reactant and cannot, therefore, shed any light on the homologation reaction sequence. The reaction and deactivation mechanism of the zeolite H‐ZSM‐5‐catalyzed ETH process was elucidated using a combination of complementary solid‐state NMR and operando UV/Vis diffuse reflectance spectroscopy, coupled with on‐line mass spectrometry. This approach establishes the existence of a homologation reaction sequence through analysis of the pattern of the identified reactive and deactivated species. Furthermore, and in contrast to the MTH process, the deficiency of any olefinic‐hydrocarbon pool species (that is, the olefin cycle) during the ETH process is also noted.

Predicting the structure and thermodynamics of protein–protein interactions (PPIs) are key to a proper understanding and modulation of their function. Since experimental methods might not be able to catch up with the fast growth of genomic data, computational alternatives are therefore required. We present here a review dealing with various aspects of predicting binding affinity changes upon mutations (ΔΔG). We focus on predictors that consider three-dimensional structure information to estimate the impact of mutations on the binding affinity of a protein–protein complex, excluding the rigorous free energy perturbation methods. Training and evaluation, ΔΔG databases, data selection, and existing ΔΔG predictors are specially emphasized. We also establish the parallel with scoring functions used in docking since those share many similar PPI features with ΔΔG predictors. The field has seen a common evolution of ΔΔG predictors and scoring functions over time, transforming from purely energetic functions to statistical energy-based and further to machine learning-based functions. As machine learning has come to age, limitations in terms of quantity, quality and variety of the available data become the bottlenecks for the future development of these computational methods. This can be alleviated by building infrastructures for data generation, collection and sharing. Further developments can be catalyzed by conducting community-wide blind challenges for method assessment. This article is categorized under: Structure and Mechanism > Molecular Structures Structure and Mechanism > Computational Biochemistry and Biophysics Molecular and Statistical Mechanics > Molecular Interactions.

Eukaryotic cells, when exposed to environmental or internal stress, activate the integrated stress response (ISR) to restore homeostasis and promote cell survival. Specific stress stimuli prompt dedicated stress kinases to phosphorylate eukaryotic initiation factor 2 (eIF2). Phosphorylated eIF2 (p-eIF2) in turn sequesters the eIF2-specific guanine exchange factor eIF2B to block eIF2 recycling, thereby halting translation initiation and reducing global protein synthesis. To circumvent stress-induced translational shutdown, viruses encode ISR antagonists. Those identified so far prevent or reverse eIF2 phosphorylation. We now describe two viral proteins-one from a coronavirus and the other from a picornavirus-that have independently acquired the ability to counteract the ISR at its very core by acting as a competitive inhibitor of p-eIF2-eIF2B interaction. This allows continued formation of the eIF2-GTP-Met-tRNAi ternary complex and unabated global translation at high p-eIF2 levels that would otherwise cause translational arrest. We conclude that eIF2 and p-eIF2 differ in their interaction with eIF2B to such effect that p-eIF2-eIF2B association can be selectively inhibited.

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 mPEG5kDa-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 mPEG5kDa-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 (AF4), 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. AF4 analysis further showed that the released curcumin was subsequently solubilized by albumin. In vitro analyses revealed that the curcumin-loaded mPEG5kDa-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 mPEG5kDa-b-p(HPMA-Bz)17.1kDa micelles in mice at 50 mg curcumin/kg showed a long circulation half-life for the micelles (t1/2 = 42 h), in line with the AF4 results. In contrast, the circulation time of curcumin was considerably shorter than that of the micelles (t1/2α = 0.11, t1/2β = 2.5 h) but ∼5 times longer than has been reported for free curcumin (t1/2α = 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.

Self-assembling peptides are an exemplary class of supramolecular biomaterials of broad biomedical utility. Mechanistic studies on the peptide self-assembly demonstrated the importance of the oligomeric intermediates towards the properties of the supramolecular biomaterials being formed. In this study, we demonstrate how the overall yield of the supramolecular assemblies are moderated through subtle molecular changes in the peptide monomers. This strategy is exemplified with a set of surfactant-like peptides (SLPs) with different β-sheet propensities and charged residues flanking the aggregation domains. By integrating different techniques, we show that these molecular changes can alter both the nucleation propensity of the oligomeric intermediates and the thermodynamic stability of the fibril structures. We demonstrate that the amount of assembled nanofibers are critically defined by the oligomeric nucleation propensities. Our findings offer guidance on designing self-assembling peptides for different biomedical applications, as well as insights into the role of protein gatekeeper sequences in preventing amyloidosis.

Src kinase activity is controlled by various mechanisms involving a coordinated movement of kinase and regulatory domains. Notwithstanding the extensive knowledge related to the backbone dynamics, little is known about the more subtle side-chain dynamics within the regulatory domains and their role in the activation process. Here, we show through experimental methyl dynamic results and predicted changes in side-chain conformational couplings that the SH2 structure of Fyn contains a dynamic network capable of propagating binding information. We reveal that binding the phosphorylated tail of Fyn perturbs a residue cluster near the linker connecting the SH2 and SH3 domains of Fyn, which is known to be relevant in the regulation of the activity of Fyn. Biochemical perturbation experiments validate that those residues are essential for inhibition of Fyn, leading to a gain of function upon mutation. These findings reveal how side-chain dynamics may facilitate the allosteric regulation of the different members of the Src kinase family.

Nucleosomes are a crucial platform for the recruitment and assembly of protein complexes that process the DNA. Mechanistic and structural in vitro studies typically rely on recombinant nucleosomes that are reconstituted using artificial, strong-positioning DNA sequences. To facilitate such studies on native, genomic nucleosomes, there is a need for methods to produce any desired DNA sequence in an efficient manner. The current methods either do not offer much flexibility in choice of sequence or are less efficient in yield and labor. Here, we show that ramified rolling circle amplification (RCA) can be used to produce milligram amounts of a genomic nucleosomal DNA fragment in a scalable, one-pot reaction overnight. The protocol is efficient and flexible in choice of DNA sequence. It yields 10-fold more product than PCR, and rivals production using plasmids. We demonstrate the approach by producing the genomic DNA from the human LIN28B locus and show that it forms functional nucleosomes capable of binding pioneer transcription factor Oct4.

Chromatin state is highly dependent on the nucleosome binding proteins. Herein, we used a multipronged approach employing biophysical and in vivo experiments to characterize the effects of Nucleosome Binding Peptides (NBPeps) on nucleosome and cell activity. We performed a series of structure-based calculations on the nucleosome surface interaction with GMIP1 (a novel NBPep generated in silico), and HMGN2 (nucleosome binding motif of HMGN2), which contains sites that bind DNA and the acid patch, and also LANA and H4pep (nucleosome binding motif of H4 histone tail) that only bind to the acidic patch. Biochemical assays shows that H4pep, but not HMGN2, GMIP1 and LANA, is highly specific for targeting the nucleosome, with important effects on the final nucleosome structure and robust in vivo effects. These findings suggest that NBPeps might have important therapeutic implications and relevance as tools for chromatin investigation.

Numerous proteins are involved in the multiple pathways of the DNA damage response network and play a key role to protect the genome from the wide variety of damages that can occur to DNA. An example of this is the structure-specific endonuclease ERCC1-XPF. This heterodimeric complex is in particular involved in nucleotide excision repair (NER), but also in double strand break repair and interstrand cross-link repair pathways. Here we review the function of ERCC1-XPF in various DNA repair pathways and discuss human disorders associated with ERCC1-XPF deficiency. We also overview our molecular and structural understanding of XPF-ERCC1.

Cell division in bacteria is initiated by the polymerization of FtsZ at midcell in a ring-like structure called the Z-ring. ZapA and other proteins assist Z-ring formation and ZapA binds ZapB, which senses the presence of the nucleoids. The FtsZ&ndash, ZapA binding interface was analyzed by chemical cross-linking mass spectrometry (CXMS) under in vitro FtsZ-polymerizing conditions in the presence of GTP. Amino acids residue K42 from ZapA was cross-linked to amino acid residues K51 and K66 from FtsZ, close to the interphase between FtsZ molecules in protofilaments. Five different cross-links confirmed the tetrameric structure of ZapA. A number of FtsZ cross-links suggests that its C-terminal domain of 55 residues, thought to be largely disordered, has a limited freedom to move in space. Site-directed mutagenesis of ZapA reveals an interaction site in the globular head of the protein close to K42. Using the information on the cross-links and the mutants that lost the ability to interact with FtsZ, a model of the FtsZ protofilament&ndash, ZapA tetramer complex was obtained by information-driven docking with the HADDOCK2.2 webserver.

The alarming rise of antimicrobial resistance requires antibiotics with unexploited mechanisms. Ideal templates could be antibiotics that target the peptidoglycan precursor lipid II, known as the bacterial Achilles heel, at an irreplaceable pyrophosphate group. Such antibiotics would kill multidrug-resistant pathogens at nanomolecular concentrations without causing antimicrobial resistance. However, due to the challenge of studying small membrane-embedded drug–receptor complexes in native conditions, the structural correlates of the pharmaceutically relevant binding modes are unknown. Here, using advanced highly sensitive solid-state NMR setups, we present a high-resolution approach to study lipid II-binding antibiotics directly in cell membranes. On the example of nisin, the preeminent lantibiotic, we show that the native antibiotic-binding mode strongly differs from previously published structures, and we demonstrate that functional hotspots correspond to plastic drug domains that are critical for the cellular adaptability of nisin. Thereby, our approach provides a foundation for an improved understanding of powerful antibiotics., Antibiotics that target the peptidoglycan precursor lipid II are promising templates for next-generation antibiotics. Here authors use solid-state NMR and monitor lipid II-binding antibiotics, such as nisin, directly in cell membranes.

Self‐assembling and molecular folding are ubiquitous in Nature: they drive the organization of systems ranging from living creatures to DNA molecules. Elucidating the complex dynamics underlying these phenomena is of crucial importance. However, a tool for the analysis of the various phenomena involved in protein/peptide aggregation is still missing. Here, an innovative software is developed and validated for the identification and visualization of b‐structuring and b‐sheet formation in both simulated systems and crystal structures of proteins and peptides. The novel software suite, dubbed Morphoscanner, is designed to identify and intuitively represent b‐structuring and b‐sheet formation during molecular dynamics trajectories, paying attention to temporary strand‐strand alignment, suboligomer formation and evolution of local order. Self‐assembling peptides (SAPs) constitute a promising class of biomaterials and an interesting model to study the spontaneous assembly of molecular systems in vitro. With the help of coarse‐grained molecular dynamics the self‐assembling of diverse SAPs is simulated into molten aggregates. When applied to these systems, Morphoscanner highlights different b‐structuring schemes and kinetics related to SAP sequences. It is demonstrated that Morphoscanner is a novel versatile tool designed to probe the aggregation dynamics of self‐assembling systems, adaptable to the analysis of differently coarsened simulations of a variety of biomolecules.

Understanding the 3-D distribution and nature of active sites in heterogeneous catalysts is critical to developing structure-function relationships. However, this is difficult to achieve in microporous materials as there is little relative z-contrast between active and inactive framework elements (e.g., Al, O, P, and Si), making them difficult to differentiate with electron microscopies. We have applied atom probe tomography (APT), currently the only nanometer-scale 3-D microscopy to offer routine light element contrast, to the methanol-to-hydrocarbons (MTH) catalyst SAPO-34, with Si as the active site, which may be present in the framework as either isolated Si species or clusters (islands) of Si atoms. 29Si solid-state NMR data on isotopically enriched and natural abundance materials are consistent with the presence of Si islands, and the APT results have been complemented with simulations to show the smallest detectable cluster size as a function of instrument spatial resolution and detector efficiency. We have identified significant Si-Si affinity in the materials, as well as clustering of coke deposited by the MTH reaction (13CH3OH used) and an affinity between Brønsted acid sites and coke. A comparison with simulations shows that the ultimate spatial resolution that can be attained by APT applied to molecular sieves is 0.5-1 nm. Finally, the observed 13C clusters are consistent with hydrocarbon pool mechanism intermediates that are preferentially located in regions of increased Brønsted acidity.

Zeolite activity and selectivity is often determined by the underlying proton and hydrogen-transfer reaction pathways. For the first time, we use single-molecule fluorescence microscopy to directly follow the real-time behavior of individual styrene-derived carbocationic species formed within zeolite ZSM-5. We find that intermittent fluorescence and remarkable photostability of carbocationic intermediates strongly depend on the local chemical environment imposed by zeolite framework and guest solvent molecules. The carbocationic stability can be additionally altered by changing para-substituent on the styrene moiety, as suggested by DFT calculations. Thermodynamically unstable carbocations are more likely to switch between fluorescent (carbocationic) and dark (neutral) states. However, the rate constants of this reversible change can significantly differ among individual carbocations, depending on their exact location in the zeolite framework. The lifetimes of fluorescent states and reversibility of the process can be additionally altered by changing the interaction between dimeric carbocations and solvated Brønsted acid sites in the MFI framework. Advanced multidimensional magic angle spinning solid-state NMR spectroscopy has been employed for the accurate structural elucidation of the reaction products during the zeolite-catalyzed dimerization of styrene in order to corroborate the single-molecule fluorescence microscopy data. This complementary approach of single-molecule fluorescence microscopy, NMR, and DFT collectively indicates that the relative stability of the carbocationic and the neutral states largely depends on the substituent and the local position of the Brønsted acid site within the zeolite framework. As a consequence, new insights into the host-guest chemistry between the zeolite and aromatics, in terms of their surface mobility and reactivity, have been obtained. ispartof: JOURNAL OF THE AMERICAN CHEMICAL SOCIETY vol:140 issue:43 pages:14195-14205 ispartof: location:United States status: published

Endocannabinoid peptides, or "pepcans," are endogenous ligands of the CB1 cannabinoid receptor. Depending on their length, they display diverse activity: For instance, the nona-peptide Pepcan-9, also known as hemopressin, is a powerful inhibitor of CB1, whereas the longer variant Pepcan-12, which extends by only three amino acid residues at the N-terminus, acts on both CB1 and CB2 as an allosteric modulator, although with diverse effects. Despite active research on their pharmacological applications, very little is known about structure-activity relationships of pepcans. Different structures have been proposed for the nona-peptide, which has also been reported to form fibrillar aggregates. This might have affected the outcome and reproducibility of bioactivity studies. In an attempt of elucidating the determinants of both biological activity and aggregation propensity of Pepcan-9 and Pepcan-12, we have performed their structure characterization in solvent systems characterized by different polarity and pH. We have found that, while disordered in aqueous environment, both peptides display helical structure in less polar environment, mimicking the proteic receptor milieu. In the case of Pepcan-9, this structure is fully consistent with the observed modulation of the CB1. For Pepcan-12, whose allosteric binding site is still unknown, the presented structure is compatible with the binding at one of the previously proposed allosteric sites on CB1. These findings open the way to structure-driven design of selective peptide modulators of CB1.

The rising incidence of antibiotic-resistant lung infections has instigated a much-needed search for new therapeutic strategies. One proposed strategy is the use of exogenous surfactants to deliver antimicrobial peptides (AMPs), like CATH-2, to infected regions of the lung. CATH-2 can kill bacteria through a diverse range of antibacterial pathways and exogenous surfactant can improve pulmonary drug distribution. Unfortunately, mixing AMPs with commercially available exogenous surfactants has been shown to negatively impact their antimicrobial function. It was hypothesized that the phosphatidylglycerol component of surfactant was inhibiting AMP function and that an exogenous surfactant, with a reduced phosphatidylglycerol composition would increase peptide mediated killing at a distal site. To better understand how surfactant lipids interacted with CATH-2 and affected its function, isothermal titration calorimetry and solid-state nuclear magnetic resonance spectroscopy as well as bacterial killing curves against Pseudomonas aeruginosa were utilized. Additionally, the wet bridge transfer system was used to evaluate surfactant spreading and peptide transport. Phosphatidylglycerol was the only surfactant lipid to significantly inhibit CATH-2 function, showing a stronger electrostatic interaction with the peptide than other lipids. Although diluting the phosphatidylglycerol content in an existing surfactant, through the addition of other lipids, significantly improved peptide function and distal killing, it also reduced surfactant spreading. A synthetic phosphatidylglycerol-free surfactant however, was shown to further improve CATH-2 delivery and function at a remote site. Based on these in vitro experiments synthetic phosphatidylglycerol-free surfactants seem optimal for delivering AMPs to the lung.

Single-stranded DNA-binding proteins (SSBs) are required for all known DNA metabolic events such as DNA replication, recombination and repair. While a wealth of structural and functional data is available on the essential human SSB, hSSB1 (NABP2/OBFC2B), the close homolog hSSB2 (NABP1/OBFC2A) remains relatively uncharacterized. Both SSBs possess a well-structured OB (oligonucleotide/oligosaccharide-binding) domain that is able to recognize single-stranded DNA (ssDNA) followed by a flexible carboxyl-tail implicated in the interaction with other proteins. Despite the high sequence similarity of the OB domain, several recent studies have revealed distinct functional differences between hSSB1 and hSSB2. In this study, we show that hSSB2 is able to recognize cyclobutane pyrimidine dimers (CPD) that form in cellular DNA as a consequence of UV damage. Using a combination of biolayer interferometry and NMR, we determine the molecular details of the binding of the OB domain of hSSB2 to CPD-containing ssDNA, confirming the role of four key aromatic residues in hSSB2 (W59, Y78, W82, and Y89) that are also conserved in hSSB1. Our structural data thus demonstrate that ssDNA recognition by the OB fold of hSSB2 is highly similar to hSSB1, indicating that one SSB may be able to replace the other in any initial ssDNA binding event. However, any subsequent recruitment of other repair proteins most likely depends on the divergent carboxyl-tail and as such is likely to be different between hSSB1 and hSSB2.

The function of chromatin ultimately depends on the many chromatin-associated proteins and protein complexes that regulate all DNA-templated processes such as transcription, repair and replication. As the molecular docking platform for these proteins, the nucleosome is the essential gatekeeper to the genome. As such, the nucleosome-binding activity of a myriad of proteins is essential for a healthy cell. Here, we review the molecular basis of nucleosome-protein interactions and classify the different binding modes available. The structural data needed for such studies not only come from traditional sources such as X-Ray crystallography but also increasingly from other sources. In particular, we highlight how partial interaction data, derived from for example NMR or mutagenesis, are used in data-driven docking to drive the modeling of the complex into an atomistic structure. This approach has opened up detailed insights for several nucleosome-protein complexes that were intractable or recalcitrant to traditional methods. These structures guide the formation of new hypotheses and advance our understanding of chromatin function at the molecular level.