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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.)

Antimicrobial peptides are a promising class of potential antibiotics that interact selectively with negatively charged lipid bilayers. This paper presents the structural characterization of the antimicrobial peptides myxinidin and WMR associated with bacterial membrane mimetic micelles and bicelles by NMR, CD spectroscopy, and molecular dynamics simulations. Both peptides adopt a different conformation in the lipidic environment than in aqueous solution. The location of the peptides in micelles and bicelles has been studied by paramagnetic relaxation enhancement experiments with paramagnetic tagged 5- and 16-doxyl stearic acid (5-/16-SASL). Molecular dynamics simulations of multiple copies of the peptides were used to obtain an atomic level of detail on membrane-peptide and peptide-peptide interactions. Our results highlight an essential role of the negatively charged membrane mimetic in the structural stability of both myxinidin and WMR. The peptides localize predominantly in the membrane's headgroup region and have a noticeable membrane thinning effect on the overall bilayer structure. Myxinidin and WMR show a different tendency to self-aggregate, which is also influenced by the membrane composition (DOPE/DOPG versus DOPE/DOPG/CL) and can be related to the previously observed difference in the ability of the peptides to disrupt different types of model membranes., 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. Published by Elsevier B.V.)

A new difference-spectroscopy method is introduced for measuring T 1 relaxation times. It is inspired by the earlier work of Freeman and Hill and eliminates the need for recording signal intensities at thermodynamic equilibrium. The new method is termed SIP-R (Split-Inversion Pulse and Recovery) and reduces the number of refinable parameters in the curve fitting process of relaxation-delay-dependent signal intensities by using two instead of the three parameters typically used in the standard inversion-recovery sequence. The SIP-R method preserves the dynamic range of measurement of the standard inversion-recovery method but converts the rise-to-maximum mathematical functionality of the recorded data into a decay-to-zero functionality. The decay-to-zero functionality renders the SIP-R sequence advantageous for inverse Laplace transformation numerical optimizations. The new technique proves to be extremely robust with respect to pulse imperfections, pulse-power changes during the pulse sequence, pulse-width miscalibrations, resonance offsets, and radiofrequency field variations. It also compensates for acoustic ring-down effects and proves reliable for measurements with inhomogeneously broadened signals up to several kilohertz linewidth. 1 H NMR experiments with methane gas at pressures up to 50 atm in toroid-cavity pressure vessel probes and in the presence of the methane-to-methanol conversion catalyst Cu-ZnO/Al 2 O 3 are used to show the usefulness of the new method for relaxation time investigations under pressure, at strong radiofrequency field gradients, and in the presence of paramagnetic materials., 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 © 2023 Elsevier Inc. All rights reserved.)

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 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 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).)

An infecting and propagating parasite relies on its innate defense system to evade the host's immune response and to survive challenges from commensal bacteria. More so for the nematode Anisakis, a marine parasite that during its life cycle encounters both vertebrate and invertebrate hosts and their highly diverse microbiotas. Although much is still unknown about how the nematode mitigates the effects of these microbiota, its antimicrobial peptides likely play an important role in its survival. We identified anisaxins, the first cecropin-like helical antimicrobial peptides originating from a marine parasite, by mining available genomic and transcriptomic data for Anisakis spp. These peptides are potent bactericidal agents in vitro, selectively active against Gram-negative bacteria, including multi-drug resistant strains, at sub-micromolar concentrations. Their interaction with bacterial membranes was confirmed by solid state NMR (ssNMR) and is highly dependent on the peptide concentration as well as peptide to lipid ratio, as evidenced by molecular dynamics (MD) simulations. MD results indicated that an initial step in the membranolytic mode of action involves membrane bulging and lipid extraction; a novel mechanism which may underline the peptides' potency. Subsequent steps include membrane permeabilization leading to leakage of molecules and eventually cell death, but without visible macroscopic damage, as shown by atomic force microscopy and flow cytometry. This membranolytic antibacterial activity does not translate to cytotoxicity towards human peripheral blood mononuclear cells (HPBMCs), which was minimal at well above bactericidal concentrations, making anisaxins promising candidates for further drug development. STATEMENT OF SIGNIFICANCE: Witnessing the rapid spread of antibiotic resistance resulting in millions of infected and dozens of thousands dying worldwide every year, we identified anisaxins, antimicrobial peptides (AMPs) from marine parasites, Anisakis spp., with potent bactericidal activity and selectivity towards multi-drug resistant Gram-negative bacteria. Anisaxins are membrane-active peptides, whose activity, very sensitive to local peptide concentrations, involves membrane bulging and lipid extraction, leading to membrane permeabilization and bacterial cell death. At the same time, their toxicity towards host cells is negligible, which is often not the case for membrane-active AMPs, therefore making them suitable drug candidates. Membrane bulging and lipid extraction are novel concepts that broaden our understanding of peptide interactions with bacterial functional structures, essential for future design of such biomaterials., 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 © 2022 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

We introduce an alternative way for spin-state selection, RODA, which yields higher sensitivity for spin systems exhibiting a TROSY effect. With RODA, the TROSY component of a doublet is recorded twice using a double acquisition scheme. RODA works by simple addition of consecutive NMR signals, and does not require any special processing. Thus, this pulse sequence element can seamlessly be integrated into existing experiments. We demonstrate the broad applicability of RODA with several systems exhibiting a TROSY effect on 15 N- 1 H, 19 F- 13 C or 1 H- 13 C moieties. Further, we show that virtual decoupling with increased sensitivity is possible in a single double acquisition experiment in situations as encountered with dissolution DNP., 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 © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The separation of daclatasvir and its R,R,R,R-enantiomer was studied by capillary electrophoresis using various randomly methylated β-CDs and the single isomer heptakis(2,6-di-O-methyl)-β-CD (2,6-DM-β-CD) as chiral selectors in an acidic background electrolyte. Opposite enantiomer migration order was observed for randomly substituted CDs compared to 2,6-DM-β-CD as well as methylated β-CDs with different composition according to the specifications of the manufacturers. HPLC and NMR analyses confirmed that the presence of a high 2,6-DM-β-CD content in the CDs enables to achieve the migration order R,R,R,R-enantiomer > daclatasvir. In contrast, products with low 2,6-DM-β-CD isomer content and/or the presence of a large amount of methylated CD isomers, in which d-glucopyranose moieties are not substituted in either position 2 or 6, displayed the opposite enantiomer migration order daclatasvir > R,R,R,R-enantiomer. The study indicated the importance of the type and composition of derivatized CDs on chiral separations in capillary electrophoresis as well as the importance of proper quality control for cyclodextrin manufacturers. Moreover, the observed migration order could be rationalized based on the composition and substitution pattern of the CDs., 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 © 2021. Published by Elsevier B.V.)

The recognition power and affinity pattern of various cyclodextrins (CD) towards the enantiomers of tetrahydrozoline (THZ) were studied using capillary electrophoresis (CE). As expected, affinity of THZ enantiomers and selectivity of recognition towards CD derivatives was strongly dependent on the cavity size and substituent type and pattern on the CD rims. Not only were the affinity strength and selectivity of recognition affected by the size of the cavity and chemistry of the CDs but also the affinity pattern. Another interesting example of opposite affinity pattern of enantiomers towards α- and β-CD was observed here. In addition, opposite affinity pattern of THZ enantiomers was seen towards β-CD and its acetylated derivatives, while methylation of β-CD did not affect the affinity pattern of THZ enantiomers. In order to get more information about structural mechanisms of the multivariate dependences mentioned above, rotating frame Overhauser enhancement spectroscopy (ROESY) and computation techniques were used. Significant differences between the structure of THZ complexes with different CDs with both methods were encountered. Good correlations between experimentally determined and computed structure of complexes, as well as between computed complex stabilities and enantiomer migration order (EMO) in CE were observed., 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 © 2021 Elsevier B.V. All rights reserved.)

The regulation of chromatin biology ultimately depends on the manipulation of its smallest subunit, the nucleosome. The proteins that bind and operate on the nucleosome do so, while their substrate is part of a polymer embedded in the dense nuclear environment. Their molecular interactions must in some way be tuned to deal with this complexity. Due to the rapid increase in the number of high-resolution structures of nucleosome-protein complexes and the increasing understanding of the cellular chromatin structure, it is starting to become clearer how chromatin factors operate in this complex environment. In this review, we analyze the current literature on the interplay between nucleosome-protein interactions and higher-order chromatin structure. We examine in what way nucleosomes-protein interactions can affect and can be affected by chromatin organization at the oligonucleosomal level. In addition, we review the characteristics of nucleosome-protein interactions that can cause phase separation of chromatin. Throughout, we hope to illustrate the exciting challenges in characterizing nucleosome-protein interactions beyond the nucleosome., 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 © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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., 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., (© 2020 The Authors.)

During a screening of cyclodextrins (CDs) as chiral selectors for the separation of daclatasvir (DCV) and its enantiomer by capillary electrophoresis (CE), an unusual phenomenon for CDs was observed, that is two peaks with a plateau in between using γ-CD as chiral selector. The same result was encountered when enantiopure DCV was injected or when analyzing a sample containing enantiopure DCV and γ-CD in a CD-free background electrolyte. Peak coalescence was observed at 45°C and at a pH above 3.5. Two peaks with a plateau were also observed for DCV stereoisomers as well as a structural analog. However, only a single peak was detected if one or both amino acid moieties of DCV were lacking. Nuclear magnetic resonance (NMR) experiments including Nuclear Overhauser effect-based methods showed that in solution DCV adopted a folded conformation in which the isopropyl side chain of the valine residues pointed toward the aromatic rings of DCV. Moreover, NMR unequivocally demonstrated the simultaneous formation of DCV-γ-CD inclusion complexes with 1:1 and 2:1 stoichiometry, which was corroborated by mass spectrometry. In both complexes, DCV also adopted a folded structure. The RSSR-diastereomer of DCV as well as an analog lacking one of the amino acid moieties also formed 1:1 and 2:1 complexes with γ-CD although a plateau was only observed in the case of the RSSR-diastereomer. As shown by CE-MS, both DCV-γ-CD complexes surprisingly comigrated as the first peak, while the second migrating peak represents non-complexed DCV., 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 © 2020. Published by Elsevier B.V.)

The small-pore framework sodium stannosilicate AV-10, chemical composition Na 2 SnSi 3 O 9 ·2H 2 O and known crystallographic structure, was synthesized by hydrothermal crystallization. This stannosilicate is built up of a three-dimensional network of corner-shared SiO 4 tetrahedra and SnO 6 octahedra. The SnO 6 sites are linked to six SiO 4 tetrahedra (Sn(6Si)) while each of the two crystallographically different SiO 4 units are connected to two SnO 6 and SiO 4 units (Si(2Si,2Sn)). This material was used as model compound for developing a solid-state MAS NMR strategy aimed on the challenges and possibilities for structural studies, particularly considering the short and medium range order to verify the connectivity of SiO 4 and SnO 6 of such compounds despite the low natural abundances of 4.68% for 29 Si and 8.59% for 119 Sn nuclei as a real challenge. 29 Si{ 119 Sn} and 119 Sn{ 29 Si} REDOR (Rotational-Echo Double-Resonance) NMR measurements after 1 H cross-polarization (CP) were carried out. The REDOR curves show a significant change after the "normal" quadratic short time evolution from which both (i) the shortest internuclear 29 Si - 119 Sn distances (and vice versa) and (ii) the number of corner-sharing SiO 4 tetrahedra around the SnO 6 octahedra (and vice versa) can be obtained. Based on these data, optimized 29 Si{ 119 Sn} and 119 Sn{ 29 Si} REPT-HMQC (Recoupled Polarization Transfer-Heteronuclear Multiple-Quantum Correlation, again after 1 H CP) experiments were implemented, which directly show those heterogroup connectivity as correlation peaks in a 2D spectrum. This information was also obtained using 2D 29 Si{ 119 Sn}-J-Coupling NMR experiments. Furthermore, 2D 29 Si INADEQUATE NMR experiments are also feasible, showing the connectivity of SiO 4 tetrahedra. The combination of REDOR, REPT-HMQC, J-Coupling and INADEQUATE experiments yielded a complete analysis of the short and medium range structure of this microporous stannosilicate, in agreement with the previously published structure obtained Ab Initio from powder X-Ray diffraction data (XRD)., 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 © 2020 Elsevier Inc. All rights reserved.)

Tetramethylsilane (TMS) is well-known as a reference standard of 1 H, 13 C and 29 Si NMR chemical shifts. In the present study, we have observed TMS molecules in gaseous and liquid solutions. In the gas phase, the shielding parameters of TMS are monitored as the functions of density when xenon and krypton are applied as the buffer gases. It permits the evaluation of shielding in an isolated TMS molecule which is determined from the measurements of frequency and available nuclear magnetic moments. Having the shielding constants of an isolated TMS molecule, it is possible to proceed with the evaluation of 1 H, 13 C and 29 Si TMS shielding in liquid state, which is extremely useful for the complete referencing of NMR spectra for protons, carbon-13 and silicon-29 nuclei. Consequently, the readings of chemical shifts and shielding parameters can be practically performed in the same experiment., 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 © 2020. Published by Elsevier Inc.)

Mesoporous material SBA-15 was functionalized with different polar and nonpolar groups: 3-aminopropyl, (SBA-15-NH 2 ), 3-isocyanatopropyl (SBA-15-NCO), 3-mercaptopropyl (SBA-15-SH), methyl (SBA-15-CH 3 ) and phenyl (SBA-15-Ph). The resulting surface grafted materials were investigated as matrices for controlled drug delivery. Anticancer agent, pemetrexed (disodium pemetrexed heptahydrate) was selected as a model drug and loaded in the unmodified and functionalized SBA-15 materials. Materials were characterized by elemental analysis, infrared spectroscopy, transmission electron microscopy, nitrogen adsorption/desorption analysis, small angle X-ray scattering, powder X-ray diffraction, solid state NMR spectroscopy and thermogravimetry. It was shown that surface modification has an impact on both encapsulated drug amount and release properties. Release experiments were performed into two media with different pH: simulated body fluid (pH = 7.4) and simulated gastric fluid (pH = 2). In general, the effect of pH was reflected by the lower release of pemetrexed under acidic conditions (pH = 2) compared to slightly alkaline saline environment (pH = 7.4). The release rate of pemetrexed from propylamine-, propylisocyanate- and phenyl-modified SBA-15 was found to be effectively controlled by intermolecular interactions as compared to that from pure SBA-15, SBA-15-SH, and SBA-15-CH 3 , that evidenced a steady and similar release. The highest release was observed for methyl-functionalized material whose hydrophobic surface accelerates the pemetrexed release. The data obtained from release studies were fitted using various kinetic models to determine the pemetrexed release mechanism and its release rate. The best correlations were found for Korsmeyer-Peppas and Higuchi models. Moreover, the theoretical three-parameter model for drug release kinetic was applied to calculate the strength of drug-support interactions. The in vitro cell study was performed on SKBR3 cancer cells and obtained results demonstrated that the modification of the mesoporous silica material by grafted polar/nonpolar groups may significantly affect the compatibility of this material with cells, drug release from this material and subsequent biological activity of PEM., Competing Interests: Declaration of competing interest No potential conflict of interest was reported by the authors., (Copyright © 2019 Elsevier B.V. All rights reserved.)

BET proteins such as BRD3 are oncogenic transcriptional coactivators. SPOP binding triggers their proteasomal degradation. In both endometrial and prostate cancers, SPOP mutations occur in the MATH domain, but with opposed influence on drug susceptibility. In prostate cancer, SPOP mutations presumably cause increased BET levels, decreasing BET inhibitor drug susceptibility. As opposed, in endometrial cancer, decreased BET levels concomitant with higher BET inhibitor drug susceptibility were observed. Here, we present the to our knowledge first co-crystal structure of SPOP and a bromodomain containing protein (BRD3). Our structural and biophysical data confirm the suggested loss-of-function in prostate cancer-associated SPOP mutants and provide mechanistic explanation. As opposed to previous literature, our data on endometrial cancer-associated SPOP mutants do not show altered binding behavior compared to wild-type SPOP, indicating a more complex regulatory mechanism. SPOP mutation screening may thus be considered a valuable personalized medicine tool for effective antitumor therapy., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

As a part of ongoing activities towards the design of ligands against pathogenic lectins, a synthesis of original α-C-galacto/α-C-manno/α-C-fucopyranosyl glycomimetics based on a calix[4]arene scaffold and their binding evaluation is described. The interactions of the glycomimetics with seven lectins of various origins were carried out using agglutination inhibition assays. The 1,3-alternate tetra-C-fucosylated ligand and its derivative having a tertBu group at the upper rim of the calix[4]arene scaffold were the most potent towards the AAL lectin family (RSL, AFL, AAL, AOL) and BC2L-C. As AFL and RSL originate from important human (Aspergillus fumigatus) and plant (Ralstonia solanacearum) pathogens, the inhibition potency of both leading structures was assessed by surface plasmon resonance. With AFL, both structures exhibited an approximately three orders of magnitude increase in affinity compared to the reference l-fucose. The role of tertBu groups as "aglycon-assisted" events was illustrated by NMR. Furthermore, both compounds showed significantly increased ability to inhibit BC2L-C (from human pathogen Burkholderia cenocepacia) cell agglutination and were able to cross-link whole B. cenocepacia cells. Although the ligands failed to significantly inhibit the agglutination activity of LecA and LecB from Pseudomonas aeruginosa, tetra-C-galactosylated calix[4]arene with tertBu groups at the upper rim of the 1,3-alternate conformation inhibited P. aeruginosa biofilm formation efficiently. This systematic and comprehensive study highlights the fact that hydrolytically stable polyvalent C-glycomimetics should be regarded as potent and selective ligands capable of acting as antiadhesive agents., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Protein tyrosine phosphatase PTPN13, also known as PTP-BL in mice, represents a large multi-domain non-transmembrane scaffolding protein that contains five consecutive PDZ domains. Here, we report the solution structures of the extended murine PTPN13 PDZ3 domain in its apo form and in complex with its physiological ligand, the carboxy-terminus of protein kinase C-related kinase-2 (PRK2), determined by multidimensional NMR spectroscopy. Both in its ligand-free state and when complexed to PRK2, PDZ3 of PTPN13 adopts the classical compact, globular D/E fold. PDZ3 of PTPN13 binds five carboxy-terminal amino acids of PRK2 via a groove located between the EB-strand and the DB-helix. The PRK2 peptide resides in the canonical PDZ3 binding cleft in an elongated manner and the amino acid side chains in position P0 and P-2, cysteine and aspartate, of the ligand face the groove between EB-strand and DB-helix, whereas the PRK2 side chains of tryptophan and alanine located in position P-1 and P-3 point away from the binding cleft. These structures are rare examples of selective class III ligand recognition by a PDZ domain and now provide a basis for the detailed structural investigation of the promiscuous interaction between the PDZ domains of PTPN13 and their ligands. They will also lead to a better understanding of the proposed scaffolding function of these domains in multi-protein complexes assembled by PTPN13 and could ultimately contribute to low molecular weight antagonists that might even act on the PRK2 signaling pathway to modulate rearrangements of the actin cytoskeleton., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The affinity pattern of terbutaline enantiomers towards various cyclodextrins was studied using capillary electrophoresis. The affinity pattern of terbutaline enantiomers was the same towards all studied cyclodextrins except heptakis(2-O-methyl-3,6-di-O-sulfo)-β-CD. Nuclear magnetic resonance spectroscopy was used for understanding of fine structural mechanisms of interactions of β-cyclodextrin and its two sulfated derivatives with the enantiomers of terbutaline. The structure of terbutaline complexes with all 3 cyclodextrins studied was different from each other. In confirmation with our earlier studies it was shown again that capillary electrophoresis represents very sensitive technique for studies of affinity patterns in cyclodextrin complexes with chiral guests. Other instrumental (e.g. NMR spectroscopy and X-ray diffraction analysis) and theoretical techniques, although very useful for obtaining the information regarding the stoichiometry, binding constants and structure of intermolecular complexes, as well as about the forces involved in selector-selectand binding and chiral recognition, may sometimes fail to properly sense those fine differences in the affinity patterns. Therefore, it is recommended to use capillary electrophoresis in order to examine correctness of affinity pattern determined for intermolecular complexes of cyclodextrins with guest molecules by other instrumental or computation techniques., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing receptor G6b-B has emerged as a key regulator of platelet homeostasis. However, it remains unclear how it mediates its effects. Tyrosine phosphorylation of ITIM and immunoreceptor tyrosine-based switch motif (ITSM) within the cytoplasmic tail of G6b-B provides a docking site for Src homology 2 domain-containing protein-tyrosine phosphatases Shp1 and Shp2, which are also critical regulators of platelet production and function. In this study, we investigate the physiological consequences of uncoupling G6b-B from Shp1 and Shp2. To address this, we generated a transgenic mouse model expressing a mutant form of G6b-B in which tyrosine residues 212 and 238 within ITIM and ITSM were mutated to phenylalanine. Mice homozygous for the mutation ( G6b-B diY/F ) were macrothrombocytopenic, as a result of the reduction in platelet production, and had large clusters of megakaryocytes and myelofibrosis at sites of hematopoiesis, similar to those observed in G6b -deficient mice and patients. Platelets from G6b-B diY/F mice were hyporesponsive to collagen, as a result of the significant reduction in the expression of the immunoreceptor tyrosine-based activation motif (ITAM)-containing collagen receptor complex GPVI-FcR γ-chain, as well as thrombin, which could be partially rescued by costimulating the platelets with adenosine diphosphate. In contrast, platelets from G6b-B diY/F , G6b KO , and megakaryocyte-specific Shp2 KO mice were hyperresponsive to antibody-mediated cross-linking of the hemi-ITAM-containing podoplanin receptor CLEC-2, suggesting that G6b-B inhibits CLEC-2-mediated platelet activation through Shp2. Findings from this study demonstrate that G6b-B must engage with Shp1 and Shp2 to mediate its regulatory effects on platelet homeostasis., (© 2018 by The American Society of Hematology.)

The migration order of the enantiomers of medetomidine in the presence of cyclodextrins studied by capillary electrophoresis in phosphate buffer, pH 2.5, depended on the cavity size and the substitution pattern of the cyclodextrins. Opposite migration order was observed in the presence of β-cyclodextrin (β-CD) and γ-cyclodextrin (γ-CD) as well as randomly sulfated β-CD (S-β-CD) and heptakis(6-O-sulfo)-β-CD (HS-β-CD). This could be rationalized by the fact that dexmedetomidine formed more stable complexes with β-CD and S-β-CD, while levomedetomidine interacted stronger with γ-CD and HS-β-CD. The structure of the complexes was derived from rotating frame nuclear Overhauser (ROESY) experiments for β-CD, γ-CD and HS-β-CD. In the case of the native CDs, the phenyl ring of medetomidine entered the cavity through the wider secondary rim of the CDs, whereas the protonated imidazole ring was positioned inside the CD cavity interacting with the sulfate groups of HS-β-CD. Furthermore, molecular dynamics calculations also suggested opposite affinities of the medetomidine enantiomers toward β-CD and γ-CD., (Copyright © 2018 Elsevier B.V. All rights reserved.)

1 H-detected solid-state NMR in combination with 1 H/ 2 D exchange steps allows for the direct identification of very strong hydrogen bonds in membrane proteins. On the example of the membrane-embedded potassium channel KcsA, we quantify the longevity of such very strong hydrogen bonds by combining time-resolved 1 H-detected solid-state NMR experiments and molecular dynamics simulations. In particular, we show that the carboxyl-side chain of the highly conserved residue Glu51 is involved in ultra-strong hydrogen bonds, which are fully-water-exposed and yet stable for weeks. The astonishing stability of these hydrogen bonds is important for the structural integrity of potassium channels, which we further corroborate by computational studies., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

The wild-type Gtf180-ΔN glucansucrase enzyme from Lactobacillus reuteri 180 was found to catalyze the α-glucosylation of the steviol glycoside rebaudioside A, using sucrose as glucosyl donor in a transglucosylation process. Structural analysis of the formed products by MALDI-TOF mass spectrometry, methylation analysis and NMR spectroscopy showed that rebaudioside A is specifically α-d-glucosylated at the steviol C-19 β-d-glucosyl moiety (55% conversion). The main product is a mono-(α1 → 6)-glucosylated derivative (RebA-G1). A series of minor products, up to the incorporation of eight glucose residues, comprise elongations of RebA-G1 with mainly alternating (α1 → 3)- and (α1 → 6)-linked glucopyranose residues. These studies were carried out in the context of a program directed to the improvement of the taste of steviol glycosides via enzymatic modification of their naturally occurring carbohydrate moieties., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Matrix proteins (MAs) play a key role in the transport of retroviral proteins inside infected cells and in the interaction with cellular membranes. In most retroviruses, retroviral MAs are N-terminally myristoylated. This modification serves as a membrane targeting signal and also as an anchor for membrane interaction. The aim of this work was to characterize the interactions anchoring retroviral MA at the plasma membrane of infected cell. To address this issue, we compared the structures and membrane affinity of the Mason-Pfizer monkey virus (M-PMV) wild-type MA with its two budding deficient double mutants, that is, T41I/T78I and Y28F/Y67F. The structures of the mutants were determined using solution NMR spectroscopy, and their interactions with water-soluble phospholipids were studied. Water-soluble phospholipids are widely used models for studying membrane interactions by solution NMR spectroscopy. However, this approach might lead to artificial results due to unnatural hydrophobic interactions. Therefore, we used a new approach based on the measurement of the loss of the 1 H NMR signal intensity of the protein sample induced by the addition of the liposomes containing phospholipids with naturally long fatty acids. HIV-1 MA was used as a positive control because its ability to interact with liposomes has already been described. We found that in contrast to HIV-1, the M-PMV MA interacted with the liposomes differently and much weaker. In our invivo experiments, the M-PMV MA did not co-localize with lipid rafts. Therefore, we concluded that M-PMV might adopt a different membrane binding mechanism than HIV-1., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

A low-cost single-crystal NMR kit is presented along with a web-based post-processing software. The kit consists of a piezo-crystal motor and a goniometer for the crystal, both embedded in a standard wide-bore NMR probe with a 3D printed scaffold. The NMR pulse program controls the angle setting automatically, and the post-processing software incorporates a range of orientation-angle discrepancies present in the kit and other single-crystal setups. Results with a NaNO3 single-crystal show a high degree of reproducibility and excellent agreement with previous findings for the anisotropic quadrupolar interaction., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Solvation and hydration are key factors for determining the stability and folding of proteins, as well as the formation of amyloid fibrils and related polypeptide aggregates. Using attenuated total reflectance Fourier-transform infrared and solid-state NMR spectroscopy, we find that the Aβ peptide experiences a remarkable conformational switch from β to α secondary structure upon solvent removal by lyophilization of oligomers. This transition is, contrary to Aβ fibrils, independent of concentration of organic co-solvents or co-solutes and is reversible upon re-addition of the solvent. Our data illuminate a previously unnoted secondary structural plasticity of the Aβ peptide in amyloid oligomers that could bear relevance for Aβ's interactions with cellular structures of low polarity., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

GTFA, a glucansucrase enzyme of the probiotic bacterium Lactobacillus reuteri 121, is capable of synthesizing an α-glucan polysaccharide with (1 → 4) and (1 → 6) linkages from sucrose. With respect to its biosynthesis, the present study has shown that the ratio of oligosaccharide versus polysaccharide synthesized was directly proportional to the concentration of sucrose. It appears that the size distribution of products is kinetically controlled, but the linkage distribution in the polysaccharide material is not changed. At high sucrose concentrations the sucrose isomers leucrose and trehalulose were synthesized, using the accumulated fructose as acceptor, together with 4'- and 6'-α-D-glucosyl-leucrose and 6'-α-D-glucosyl-trehalulose. The finding of an additional branched hexasaccharide demonstrates that the enzyme is able to introduce branch-points already in relatively short oligosaccharides., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Different fungi, including the genera Neosartorya, Byssochlamys and Talaromyces, produce (asco)spores that survive pasteurization treatments and are regarded as the most stress-resistant eukaryotic cells. Here, the NMR analysis of a series of trehalose-based oligosaccharides, being compatible solutes that are accumulated to high levels in ascospores of the fungus Neosartorya fischeri, is presented. These oligosaccharides consist of an α,α-trehalose backbone, extended with one [α-D-Glcp-(1 → 6)-α-D-Glcp-(1 ↔ 1)-α-D-Glcp; isobemisiose], two [α-D-Glcp-(1 → 6)-α-D-Glcp-(1 → 6)-α-D-Glcp-(1 ↔ 1)-α-D-Glcp] or three [α-D-Glcp-(1 → 6)-α-D-Glcp-(1 → 6)-α-D-Glcp-(1 → 6)-α-D-Glcp-(1 ↔ 1)-α-D-Glcp] glucose units. The tetra- and pentasaccharide, dubbed neosartose and fischerose, respectively, have not been reported before to occur in nature., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The outer membrane protein BamA is the key player in β-barrel assembly in Gram-negative bacteria. Despite the availability of high-resolution crystal structures, the dynamic behavior of the transmembrane domain and the large periplasmic extension consisting of five POTRA (POlypeptide-TRansport-Associated) domains remains unclear. We demonstrate reconstitution of full-length BamA in proteoliposomes at low lipid-to-protein ratio, leading to high sensitivity and resolution in solid-state NMR (ssNMR) experiments. We detect POTRA domains in ssNMR experiments probing rigid protein segments in our preparations. These results suggest that the periplasmic region of BamA is firmly attached to the β-barrel and does not experience fast global motion around the angle between POTRA 2 and 3. We show that this behavior holds at lower protein concentrations and elevated temperatures. Chemical shift variations observed after reconstitution in lipids with different chain lengths and saturation levels are compatible with conformational plasticity of BamA's transmembrane domain. Electron microscopy of the ssNMR samples shows that BamA can cause local disruptions of the lipid bilayer in proteoliposomes. The observed interplay between protein-protein and protein-lipid interactions may be critical for BamA-mediated insertion of substrates into the outer membrane., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Availability of high-resolution atomic structures is one of the prerequisites for a mechanistic understanding of biomolecular function. This atomic information can, however, be difficult to acquire for interesting systems such as high molecular weight and multi-subunit complexes. For these, low-resolution and/or sparse data from a variety of sources including NMR are often available to define the interaction between the subunits. To make best use of all the available information and shed light on these challenging systems, integrative computational tools are required that can judiciously combine and accurately translate the sparse experimental data into structural information. In this Perspective we discuss NMR techniques and data sources available for the modeling of large and multi-subunit complexes. Recent developments are illustrated by particularly challenging application examples taken from the literature. Within this context, we also position our data-driven docking approach, HADDOCK, which can integrate a variety of information sources to drive the modeling of biomolecular complexes. It is the synergy between experimentation and computational modeling that will provides us with detailed views on the machinery of life and lead to a mechanistic understanding of biomolecular function., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

Two different NMR pulse schemes involving sequential (1)H data acquisition are presented for achieving protein backbone sequential resonance assignments: (i) acquisition of 3D {HCCNH and HNCACONH} and (ii) collection of 3D {HNCOCANH and HNCACONH} chemical shift correlation spectra using uniformly (13)C,(15)N labelled proteins. The sequential acquisition of these spectra reduces the overall experimental time by a factor of ≈2 as compared to individual acquisitions. The suitability of this approach is experimentally demonstrated for the C-terminal winged helix (WH) domain of the minichromosome maintenance (MCM) complex of Sulfolobus solfataricus., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Mobile and cost-effective NMR spectroscopy exploiting low-field permanent magnets is a field of tremendous development with obvious applications for arrayed large scale analysis, field work, and industrial screening. So far such demonstrations have concentrated on relaxation measurements and lately high-resolution liquid-state NMR applications. With high-resolution solid-state NMR spectroscopy being increasingly important in a broad variety of applications, we here introduce low-field magic-angle spinning (MAS) solid-state multinuclear NMR based on a commercial ACT 0.45 T 62 mm bore Halbach magnet along with a homebuilt FPGA digital NMR console, amplifiers, and a modified standard 45 mm wide MAS probe for 7 mm rotors. To illustrate the performance of the instrument and address cases where the low magnetic field may offer complementarity to high-field NMR experiments, we demonstrate applications for (23)Na MAS NMR with enhanced second-order quadrupolar coupling effects and (31)P MAS NMR where reduced influence from chemical shift anisotropy at low field may facilitate determination of heteronuclear dipole-dipole couplings., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The highly viscous neutral exopolysaccharide, produced in large amounts by Lactobacillus fermentum TDS030603 in MRS broth and in a chemically defined medium supplemented with glucose, was investigated by GLC(-EIMS) monosaccharide and methylation analysis, periodate oxidation, MALDI-TOF mass spectrometry, and 1D/2D NMR spectroscopy. The GLC(-EIMS) analyses showed that the EPS contained d-glucose and d-galactose in an averaged molar ratio of 2.6:1.0, consisting of terminal d-Glcp, 3-substituted d-Glcp, 2,3-disubstituted d-Glcp, and 6-substituted d-Galp in an averaged molar ratio of 1.3:1.0:1.1:1.1, with a trace of terminal d-Galp (0.1). Combined with the NMR data of the EPS (previously reported constituent analysis: terminal Glcp, 3-substituted Glcp, 2,3-disubstituted Glcp, and 6-substituted Galp in an averaged molar ratio of 1.2:1.1:1.0:1.1) and oligosaccharides isolated after partial acid hydrolysis, a branched tetrasaccharide repeating unit, with some heterogeneity in the side chain, having the following structure is proposed: [fromula see text]., (Copyright © 2013 Elsevier Ltd. All rights reserved.)