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Liquid-liquid phase separation is the key process underlying formation of membrane-less compartments in cells. A highly dynamic cellular body with rapid component exchange is Cajal body (CB), which supports the extensive compositional dynamics of the RNA splicing machinery, spliceosome. Here, we select an arginine-glycine (RG)-rich segment of coilin, the major component of CB, establish its RNA-induced phase separation, and through combined use of nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) probes, interrogate its dynamics within the crowded interior of formed droplets. Taking advantage of glycine-based singlet-states, we show that glycines retain a large level of sub-nanoseconds dynamics inside the coilin droplets. Furthermore, the continuous-wave (CW) and electron-electron dipolar (PELDOR) and electron-nucleus hyperfine coupling EPR data (HYSCORE) support the RNA-induced formation of dynamic coilin droplets with high coilin peptide concentrations. The combined NMR and EPR data reveal the high dynamics of the RG-rich coilin within droplets and suggest its potential role in the large dynamics of CBs. The authors show the RNA-induced droplet formation by a component of Cajal bodies and reveal the large nanoseconds mobility of glycine residues inside droplets as a molecular factor potentially contributing to the large dynamics of Cajal bodies. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is a progressive and incurable neurodegenerative disease characterized by the extracellular deposition of amyloid plaques. Investigation into the composition of these plaques revealed a high amount of amyloid-β (Aβ) fibrils and a high concentration of lipids, suggesting that fibril-lipid interactions may also be relevant for the pathogenesis of AD. Therefore, we grew Aβ40 fibrils in the presence of lipid vesicles and determined their structure by cryo-electron microscopy (cryo-EM) to high resolution. The fold of the major polymorph is similar to the structure of brain-seeded fibrils reported previously. The majority of the lipids are bound to the fibrils, as we show by cryo-EM and NMR spectroscopy. This apparent lipid extraction from vesicles observed here in vitro provides structural insights into potentially disease-relevant fibril-lipid interactions. Alzheimer's plaques contain a high amount of Aβ fibrils and a high concentration of lipids. The authors determined structures of Aβ40 fibrils grown in the presence of lipids, revealing high-resolution details of potentially disease-relevant fibril-lipid interactions. [ABSTRACT FROM AUTHOR]

Anisotropic NMR spectroscopy, revealing residual dipolar couplings (RDCs) and residual chemical shift anisotropies (RCSAs) has emerged as a powerful tool to determine the configurations of synthetic and complex natural compounds. The deduction of the absolute in addition to the relative configuration is one of the primary goals in the field. Therefore, the investigation of the enantiodiscriminating capabilities of chiral alignment media becomes essential. While RDCs and RCSAs are now used for the determination of the relative configuration routinely, RCSAs have not been measured in chiral alignment media such as chiral liquid crystals. Herein, we present this application by measuring RCSAs for chiral analytes such as indanol and isopinocampheol in the lyotropic liquid crystalline phase of an L‐valine derived helically chiral polyacetylenes. We have also demonstrated that a single 1D 13C−{1H} NMR spectrum suffices to get the RCSAs circumventing the necessity to acquire two spectra at two alignment conditions. [ABSTRACT FROM AUTHOR]

Anisotropic NMR spectroscopy, revealing residual dipolar couplings (RDCs) and residual chemical shift anisotropies (RCSAs) has emerged as a powerful tool to determine the configurations of synthetic and complex natural compounds. The deduction of the absolute in addition to the relative configuration is one of the primary goals in the field. Therefore, the investigation of the enantiodiscriminating capabilities of chiral alignment media becomes essential. While RDCs and RCSAs are now used for the determination of the relative configuration routinely, RCSAs have not been measured in chiral alignment media such as chiral liquid crystals. Herein, we present this application by measuring RCSAs for chiral analytes such as indanol and isopinocampheol in the lyotropic liquid crystalline phase of an L‐valine derived helically chiral polyacetylenes. We have also demonstrated that a single 1D 13C−{1H} NMR spectrum suffices to get the RCSAs circumventing the necessity to acquire two spectra at two alignment conditions. [ABSTRACT FROM AUTHOR]

An in‐depth structural characterization of marine dissolved organic matter (DOM) is crucial for a better understanding of its connection to marine and global biogeochemical cycles. High‐field nuclear magnetic resonance (NMR) spectroscopy in general and two‐dimensional (2D) correlation spectroscopy (COSY) in particular are powerful tools for the molecular level structural analysis of marine DOM. These 2D NMR experiments demand prolonged experimental times of days to weeks per sample due to the requirement of a large number of experiments to record the second dimension (t1) of the 2D NMR experiment. Herein, we demonstrate the efficacy of nonuniform sampling (NUS) in 2D COSY, which (i) reduces the measurement time by half without compromising spectral quality and (ii) enhances the signal intensity for the given experiment time. This approach can lead to substantial progress in the structural analysis of previously poorly characterized marine DOM. NUS COSY has been exemplified on two solid‐phase extracted DOM samples from the surface and deep ocean at 800 MHz and 1.2 GHz instruments. A dramatic improvement in sensitivity and spectral resolution is observed in NUS COSY spectra recorded at 1.2 GHz instrument when compared to 800 MHz instrument. NUS COSY NMR is versatile and anticipated to have significant potential for uncovering the hidden molecular diversity of DOM from various aquatic environments within a reasonable timeframe. The introduction of NUS into the environmental sciences was long overdue, and our study now opens the door for a wide field of new applications of NMR in the marine and aquatic sciences. [ABSTRACT FROM AUTHOR]

The previously undescribed natural product lumnitzeralactone (1), which represents a derivative of ellagic acid, was isolated from the anti-bacterial extract of the Indonesian mangrove species Lumnitzera racemosa Willd. The structure of lumnitzeralactone (1), a proton-deficient and highly challenging condensed aromatic ring system, was unambiguously elucidated by extensive spectroscopic analyses involving high-resolution mass spectrometry (HRMS), 1D 1H and 13C nuclear magnetic resonance spectroscopy (NMR), and 2D NMR (including 1,1-ADEQUATE and 1,n-ADEQUATE). Determination of the structure was supported by computer-assisted structure elucidation (CASE system applying ACD-SE), density functional theory (DFT) calculations, and a two-step chemical synthesis. Possible biosynthetic pathways involving mangrove-associated fungi have been suggested. [ABSTRACT FROM AUTHOR]

α-synuclein misfolding and aggregation into fibrils is a common feature of α-synucleinopathies, such as Parkinson's disease, in which α-synuclein fibrils are a characteristic hallmark of neuronal inclusions called Lewy bodies. Studies on the composition of Lewy bodies extracted postmortem from brain tissue of Parkinson's patients revealed that lipids and membranous organelles are also a significant component. Interactions between α-synuclein and lipids have been previously identified as relevant for Parkinson's disease pathology, however molecular insights into their interactions have remained elusive. Here we present cryo-electron microscopy structures of six α-synuclein fibrils in complex with lipids, revealing specific lipid-fibril interactions. We observe that phospholipids promote an alternative protofilament fold, mediate an unusual arrangement of protofilaments, and fill the central cavities of the fibrils. Together with our previous studies, these structures also indicate a mechanism for fibril-induced lipid extraction, which is likely to be involved in the development of α-synucleinopathies. Specifically, one potential mechanism for the cellular toxicity is the disruption of intracellular vesicles mediated by fibrils and oligomers, and therefore the modulation of these interactions may provide a promising strategy for future therapeutic interventions. Interactions between α-synuclein fibrils and lipids have been associated with the development of Parkinson's disease. This cryo-EM study reveals structural details of these interactions and suggests a mechanism for fibril-induced lipid extraction. [ABSTRACT FROM AUTHOR]

Heparins and heparan sulfate polysaccharides are negatively charged glycosaminoglycans and play important roles in cell-to-matrix and cell-to-cell signaling processes. Metal ion binding to heparins alters the conformation of heparins and influences their function. Various experimental techniques have been used to investigate metal ion-heparin interactions, frequently with inconsistent results. Exploiting the quadrupolar 23Na nucleus, we herein develop a 23Na NMR-based competition assay and monitor the binding of divalent Ca2+ and Mg2+ and trivalent Al3+ metal ions to sodium heparin and the consequent release of sodium ions from heparin. The 23Na spin relaxation rates and translational diffusion coefficients are utilized to quantify the metal ion-induced release of sodium ions from heparin. In the case of the Al3+ ion, the complementary approach of 27Al quadrupolar NMR is employed as a direct probe of ion binding to heparin. Our NMR results demonstrate at least two metal ion-binding sites with different affinities on heparin, potentially undergoing dynamic exchange. For the site with lower metal ion binding affinity, the order of Ca2+ > Mg2+ > Al3+ is obtained, in which even the weakly binding Al3+ ion is capable of displacing sodium ions from heparin. Overall, the multinuclear quadrupolar NMR approach employed here can monitor and quantify metal ion binding to heparin and capture different modes of metal ion-heparin binding. [ABSTRACT FROM AUTHOR]

Aggregation of amyloidogenic proteins is a characteristic of multiple neurodegenerative diseases. Atomic resolution of small molecule binding to such pathological protein aggregates is of interest for the development of therapeutics and diagnostics. Here we investigate the interaction between α-synuclein fibrils and anle138b, a clinical drug candidate for disease modifying therapy in neurodegeneration and a promising scaffold for positron emission tomography tracer design. We used nuclear magnetic resonance spectroscopy and the cryogenic electron microscopy structure of α-synuclein fibrils grown in the presence of lipids to locate anle138b within a cavity formed between two β-strands. We explored and quantified multiple binding modes of the compound in detail using molecular dynamics simulations. Our results reveal stable polar interactions between anle138b and backbone moieties inside the tubular cavity of the fibrils. Such cavities are common in other fibril structures as well. Understanding how small molecules bind to pathological aggregates is of importance for therapeutic and diagnostic development in diseases such as Parkinson's Disease. Here, the authors reveal a binding site of anle138b to lipid-induced α-synuclein fibrils. [ABSTRACT FROM AUTHOR]

Recent studies revealed that molecular events related with the physiology and pathology of αS might be regulated by specific sequence motifs in the primary sequence of αS. The importance of individual residues in these motifs remains an important open avenue of investigation. In this work, we have addressed the structural details related to the amyloid fibril assembly and lipid‐binding features of αS through the design of site‐directed mutants at position 39 of the protein and their study by in vitro and in vivo assays. We demonstrated that aromaticity at position 39 of αS primary sequence influences strongly the aggregation properties and the membrane‐bound conformations of the protein, molecular features that might have important repercussions for the function and dysfunction of αS. Considering that aggregation and membrane damage is an important driver of cellular toxicity in amyloid diseases, future work is needed to link our findings with studies based on toxicity and neuronal cell death. Brief statement outlining significance: Modulation by distinct sequential motifs and specific residues of αS on its physiological and pathological states is an active area of research. Here, we demonstrated that aromaticity at position 39 of αS modulates the membrane‐bound conformations of the protein, whereas removal of aromatic functionality at position 39 reduces strongly the amyloid assembly in vitro and in vivo. Our study provides new evidence for the modulation of molecular events related with the physiology and pathology of αS. [ABSTRACT FROM AUTHOR]

Nuclear magnetic resonance (NMR) is widely applied from analytics to biomedicine although it is an inherently insensitive phenomenon. Overcoming sensitivity challenges is key to further broaden the applicability of NMR and, for example, improve medical diagnostics. Here, we present a rapid strategy to enhance the signals of 13C‐labelled metabolites with para‐hydrogen and, in particular, 13C‐pyruvate, an important molecule for the energy metabolism. We succeeded to obtain an average of 27 % 13C polarization of 1‐13C‐pyruvate in water which allowed us to introduce two applications for studying cellular metabolism. Firstly, we demonstrate that the metabolism of 1‐13C‐pyruvate can serve as a biomarker in cellular models of Parkinson's disease and, secondly, we introduce the opportunity to combine real‐time metabolic analysis with protein structure determination in the same cells. Based on the here presented results, we envision the use of our approach for future biomedical studies to detect diseases. [ABSTRACT FROM AUTHOR]

Partner recognition in protein binding is critical for all biological functions, and yet, delineating its mechanism is challenging, especially when recognition happens within microseconds. We present a theoretical and experimental framework based on straight-forward nuclear magnetic resonance relaxation dispersion measurements to investigate protein binding mechanisms on sub-millisecond timescales, which are beyond the reach of standard rapid-mixing experiments. This framework predicts that conformational selection prevails on ubiquitin's paradigmatic interaction with an SH3 (Src-homology 3) domain. By contrast, the SH3 domain recognizes ubiquitin in a two-state binding process. Subsequent molecular dynamics simulations and Markov state modeling reveal that the ubiquitin conformation selected for binding exhibits a characteristically extended C-terminus. Our framework is robust and expandable for implementation in other binding scenarios with the potential to show that conformational selection might be the design principle of the hubs in protein interaction networks. The authors provide a litmus test for the recognition mechanism of transiently binding proteins based on nuclear magnetic resonance and find a conformational selection binding mechanism through concentration-dependent kinetics of ubiquitin and SH3. [ABSTRACT FROM AUTHOR]

The seaweed Sargassum muticum, collected on the southern coast of Galicia, yielded a tetraprenyltoluquinol chromane meroditerpene compound known as 1b, whose structure is revised. The relative configuration of 1b was determined by J-based configurational methodology combined with an iJ/DP4 statistical analysis and further confirmed by measuring two anisotropic properties: carbon residual chemical shift anisotropies (13C-RCSAs) and one-bond 1H-13C residual dipolar couplings (1DCH-RDCs). The absolute configuration of 1b was deduced by ECD/OR/TD-DFT methods and established as 3R,7S,11R. [ABSTRACT FROM AUTHOR]

Three new diterpene alkaloids, (+)-8-epiagelasine T (1), (+)-10-epiagelasine B (2), and (+)-12-hydroxyagelasidine C (3), along with three known compounds, (+)-ent-agelasine F (4), (+)-agelasine B (5), and (+)-agelasidine C (6), were isolated from the sponge Agelas citrina, collected on the coasts of the Yucatán Peninsula (Mexico). Their chemical structures were elucidated by 1D and 2D NMR spectroscopy, HRESIMS techniques, and a comparison with literature data. Although the synthesis of (+)-ent-agelasine F (4) has been previously reported, this is the first time that it was isolated as a natural product. The evaluation of the antimicrobial activity against the Gram-positive pathogens Staphylococcus aureus, Streptococcus pneumoniae, Enterococcus faecalis showed that all of them were active, with (+)-10-epiagelasine B (2) being the most active compound with an MIC in the range of 1–8 µg/mL. On the other hand, the Gram-negative pathogenes Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae were also evaluated, and only (+)-agelasine B (5) showed a moderate antibacterial activity with a MIC value of 16 μg/mL. [ABSTRACT FROM AUTHOR]

In a wide spectrum of neurodegenerative diseases, self-assembly of pathogenic proteins to cytotoxic intermediates is accelerated by the presence of metal ions such as Cu2+. Only low concentrations of these early transient oligomeric intermediates are present in a mixture of species during fibril formation, and hence information on the extent of structuring of these oligomers is still largely unknown. Here, we investigate dimers as the first intermediates in the Cu2+-driven aggregation of a cyclic D , L -α-peptide architecture. The unique structural and functional properties of this model system recapitulate the self-assembling properties of amyloidogenic proteins including β-sheet conformation and cross-interaction with pathogenic amyloids. We show that a histidine-rich cyclic D , L -α-octapeptide binds Cu2+ with high affinity and selectivity to generate amyloid-like cross-β-sheet structures. By taking advantage of backbone amide methylation to arrest the self-assembly at the dimeric stage, we obtain structural information and characterize the degree of local order for the dimer. We found that, while catalytic amounts of Cu2+ promote aggregation of the peptide to fibrillar structures, higher concentrations dose-dependently reduce fibrillization and lead to formation of spherical particles, showing self-assembly to different polymorphs. For the initial self-assembly step to the dimers, we found that Cu2+ is coordinated on average by two histidines, similar to self-assembled peptides, indicating that a similar binding interface is perpetuated during Cu2+-driven oligomerization. The dimer itself is found in heterogeneous conformations that undergo dynamic exchange, leading to the formation of different polymorphs at the initial stage of the aggregation process. [ABSTRACT FROM AUTHOR]

Cohesive FG domains assemble into a condensed phase forming the selective permeability barrier of nuclear pore complexes. Nanoscopic insight into fundamental cohesive interactions has long been hampered by the sequence heterogeneity of native FG domains. We overcome this challenge by utilizing an engineered perfectly repetitive sequence and a combination of solution and magic angle spinning NMR spectroscopy. We map the dynamics of cohesive interactions in both phase-separated and soluble states at atomic resolution using TROSY for rotational correlation time (TRACT) measurements. We find that FG repeats exhibit nanosecond-range rotational correlation times and remain disordered in both states, although FRAP measurements show slow translation of phase-separated FG domains. NOESY measurements enable the direct detection of contacts involved in cohesive interactions. Finally, increasing salt concentration and temperature enhance phase separation and decrease local mobility of FG repeats. This lower critical solution temperature (LCST) behaviour indicates that cohesive interactions are driven by entropy. The permeability barrier of nuclear pores is formed by disordered and yet self-interacting FG repeat domains, whose sequence heterogeneity is a challenge for mechanistic insights. Here the authors overcome this challenge and characterize the protein's dynamics by applying NMR techniques to an FG phase system that has been simplified to its essentials. [ABSTRACT FROM AUTHOR]

NMR thermometers are a convenient way to determine the temperature inside the sample of an NMR spectrometer. They rely on signals with strongly temperature‐dependent chemical shifts, often of OH groups; 99.8% perdeuterated methanol is an established example which is particularly well suited for modern, high‐sensitivity spectrometers, but it is so far calibrated only in the range of 282 to 330 K. In this work, we extend this calibration to the entire liquid range of methanol, 175 to 338 K. Additionally, we use a temperature sensor calibrated traceably to the International Temperature Scale (ITS‐90) and accounted for the magnetic field effect on the sensor, yielding a more accurate calibration curve with an uncertainty (2σ) varying between 25 and 190 mK. [ABSTRACT FROM AUTHOR]

Tony Keller, a pioneer in the field of Nuclear Magnetic Resonance (NMR) spectroscopy, passed away on October 27, 2023, at the age of 86 in Spiez, Switzerland. His work and vision were essential to the development and commercialization of NMR spectrometers for many areas of scientific research. Keller's contributions include the development of the world's first multinuclear, Fourier transform NMR spectrometer and the introduction of proton‐broadband decoupling in Fourier‐transform mode, which revolutionized heteronuclear NMR spectroscopy. He also played a significant role in the advancement of NMR technology, including the creation of superconducting magnets, digital spectrometers, cryogenically cooled probes, and high‐field NMR spectrometers. Keller's legacy will continue to inspire future generations of scientists. [Extracted from the article]

Tony Keller, a pioneer in the field of Nuclear Magnetic Resonance (NMR) spectroscopy, passed away at the age of 86 in Spiez, Switzerland. He developed the world's first multinuclear, Fourier transform NMR spectrometer and revolutionized heteronuclear NMR spectroscopy. Keller's visionary approach led to groundbreaking technologies such as superconducting magnets, digital spectrometers, cryogenically cooled probes, and high‐field NMR spectrometers. He was known for his humility, respectfulness, and contributions to science. [Extracted from the article]

Nuclear magnetic resonance is usually drastically limited by its intrinsically low sensitivity: Only a few spins contribute to the overall signal. To overcome this limitation, hyperpolarization methods were developed that increase signals several times beyond the normal/thermally polarized signals. The ideal case would be a universal approach that can signal enhance the complete sample of interest in solution to increase detection sensitivity. Here, we introduce a combination of para‐hydrogen enhanced magnetic resonance with the phenomenon of the RASER: Large signals of para‐hydrogen enhanced molecules interact with the magnetic resonance coil in a way that the signal is spontaneously converted into an in‐phase signal. These molecules directly interact with other compounds via dipolar couplings and enhance their signal. We demonstrate that this is not only possible for solvent molecules but also for an amino acid. [ABSTRACT FROM AUTHOR]

Nuclear magnetic resonance is usually drastically limited by its intrinsically low sensitivity: Only a few spins contribute to the overall signal. To overcome this limitation, hyperpolarization methods were developed that increase signals several times beyond the normal/thermally polarized signals. The ideal case would be a universal approach that can signal enhance the complete sample of interest in solution to increase detection sensitivity. Here, we introduce a combination of para‐hydrogen enhanced magnetic resonance with the phenomenon of the RASER: Large signals of para‐hydrogen enhanced molecules interact with the magnetic resonance coil in a way that the signal is spontaneously converted into an in‐phase signal. These molecules directly interact with other compounds via dipolar couplings and enhance their signal. We demonstrate that this is not only possible for solvent molecules but also for an amino acid. [ABSTRACT FROM AUTHOR]

Thirty-five peptides selected from functionally-relevant SARS-CoV-2 spike (S), membrane (M), and envelope (E) proteins were suitably modified for immunising MHC class II (MHCII) DNA-genotyped Aotus monkeys and matched with HLA-DRβ1* molecules for use in humans. This was aimed at producing the first minimal subunit-based, chemically-synthesised, immunogenic molecules (COLSARSPROT) covering several HLA alleles. They were predicted to cover 48.25% of the world's population for 6 weeks (short-term) and 33.65% for 15 weeks (long-lasting) as they induced very high immunofluorescent antibody (IFA) and ELISA titres against S, M and E parental native peptides, SARS-CoV-2 neutralising antibodies and host cell infection. The same immunological methods that led to identifying new peptides for inclusion in the COLSARSPROT mixture were used for antigenicity studies. Peptides were analysed with serum samples from patients suffering mild or severe SARS-CoV-2 infection, thereby increasing chemically-synthesised peptides' potential coverage for the world populations up to 62.9%. These peptides' 3D structural analysis (by 1H-NMR acquired at 600 to 900 MHz) suggested structural-functional immunological association. This first multi-protein, multi-epitope, minimal subunit-based, chemically-synthesised, highly immunogenic peptide mixture highlights such chemical synthesis methodology's potential for rapidly obtaining very pure, highly reproducible, stable, cheap, easily-modifiable peptides for inducing immune protection against COVID-19, covering a substantial percentage of the human population. [ABSTRACT FROM AUTHOR]

A transporter of the multidrug and toxic compound extrusion (MATE) family, Nicotiana tabacum MATE2 (NtMATE2), is located in the vacuole membrane of the tobacco plant root and is involved in the transportation of nicotine, a secondary or specialized metabolic compound in Solanaceae. Here, we report the crystal structures of NtMATE2 in its outward‐facing forms. The overall structure has a bilobate V‐shape with pseudo‐symmetrical assembly of the N‐ and C‐lobes. In one crystal structure, the C‐lobe cavity of NtMATE2 interacts with an unidentified molecule that may partially mimic a substrate. In addition, NtMATE2‐specific conformational transitions imply that an unprecedented movement of the transmembrane α‐helix 7 is related to the release of the substrate into the vacuolar lumen. [ABSTRACT FROM AUTHOR]

α-synuclein (αSyn) is the main protein component of Lewy bodies, intracellular inclusions found in the brain of Parkinson's disease (PD) patients. Neurotoxic αSyn species are broadly modified post-translationally and, in patients with genetic forms of PD, carry genetically encoded amino acid substitutions. Mutations and C-terminal truncation can increase αSyn oligomerization and fibrillization. Although several genetic mouse models based on αSyn mutations and/or truncations exist, there is still a lack of mouse models for synucleinopathies not relying on overexpression. We report here two synucleinopathy mouse models, which are based on a triple alanine to proline mutation and a C-terminal truncation of αSyn, but do not overexpress the mutant protein when compared to the endogenous mouse protein. We knocked h αSyn TP or h αSynΔ119 (h stands for "human") into the murine αSyn locus. hαSynTP is a structure-based mutant with t riple alanine to p roline substitutions that favors oligomers, is neurotoxic and evokes PD-like symptoms in Drosophila melanogaster. hαSynΔ119 lacks 21 amino acids at the C-terminus, favors fibrillary aggregates and occurs in PD. Knocking-in of h αSyn TP or h αSynΔ119 into the murine αSyn locus places the mutant protein under the control of the endogenous regulatory elements while simultaneously disrupting the mαSyn gene. Mass spectrometry revealed that h αSyn TP and h αSynΔ119 mice produced 12 and 10 times less mutant protein, compared to mαSyn in wild type mice. We show phenotypes in 1 and 1.5 years old hαSyn TP and hαSynΔ119 mice, despite the lower levels of hαSynTP and hαSynΔ119 expression. Direct comparison of the two mouse models revealed many commonalities but also aspects unique to each model. Commonalities included strong immunoactive state, impaired olfaction and motor coordination deficits. Neither model showed DAergic neuronal loss. Impaired climbing abilities at 1 year of age and a deviant gait pattern at 1.5 years old were specific for hαSynΔ119 mice, while a compulsive behavior was exclusively detected in hαSyn TP mice starting at 1 year of age. We conclude that even at very moderate levels of expression the two αSyn variants evoke measurable and progressive deficiencies in mutant mice. The two transgenic mouse models can thus be suitable to study αSyn-variant-based pathology in vivo and test new therapeutic approaches. [ABSTRACT FROM AUTHOR]

Purpose: Deposition of misfolded alpha-synuclein (αSYN) aggregates in the human brain is one of the major hallmarks of synucleinopathies. However, a target-specific tracer to detect pathological aggregates of αSYN remains lacking. Here, we report the development of a positron emission tomography (PET) tracer based on anle138b, a compound shown to have therapeutic activity in animal models of neurodegenerative diseases. Methods: Specificity and selectivity of [3H]MODAG-001 were tested in in vitro binding assays using recombinant fibrils. After carbon-11 radiolabeling, the pharmacokinetic and metabolic profile was determined in mice. Specific binding was quantified in rats, inoculated with αSYN fibrils and using in vitro autoradiography in human brain sections of Lewy body dementia (LBD) cases provided by the Neurobiobank Munich (NBM). Results: [3H]MODAG-001 revealed a very high affinity towards pure αSYN fibrils (Kd = 0.6 ± 0.1 nM) and only a moderate affinity to hTau46 fibrils (Kd = 19 ± 6.4 nM) as well as amyloid-β1–42 fibrils (Kd = 20 ± 10 nM). [11C]MODAG-001 showed an excellent ability to penetrate the mouse brain. Metabolic degradation was present, but the stability of the parent compound improved after selective deuteration of the precursor. (d3)-[11C]MODAG-001 binding was confirmed in fibril-inoculated rat striata using in vivo PET imaging. In vitro autoradiography showed no detectable binding to aggregated αSYN in human brain sections of LBD cases, most likely, because of the low abundance of aggregated αSYN against background protein. Conclusion: MODAG-001 provides a promising lead structure for future compound development as it combines a high affinity and good selectivity in fibril-binding assays with suitable pharmacokinetics and biodistribution properties. [ABSTRACT FROM AUTHOR]

The conformation in solution of monocrotaline, a pyrrolizidine alkaloid presenting an eleven‐membered macrocyclic diester ring, has been investigated using a combination of isotropic and anisotropic nuclear magnetic resonance parameters measured in four solvents of different polarity (D2O, DMSO‐d6, CDCl3, and C6D6). Anisotropic nuclear magnetic resonance parameters were measured in different alignment media, based on their compatibility with the solvent of interest: cromoglycate liquid crystal solution was used for D2O, whereas a poly (methyl methacrylate) polymer gel was chosen for CDCl3 and C6D6, and a poly (hydroxyethyl methacrylate) gel for DMSO‐d6. Whereas the pyrrolizidine ring shows an E6exo‐puckered conformation in all of the solvents, the macrocyclic eleven‐membered ring adopts different populations of syn‐parallel and anti‐parallel relative orientation of the carbonyl groups according to the polarity of the solvent. [ABSTRACT FROM AUTHOR]

The voltage-dependent anion channel (VDAC) is one of the most highly abundant proteins found in the outer mitochondrial membrane, and was one of the earliest discovered. Here we review progress in understanding VDAC function with a focus on its structure, discussing various models proposed for voltage gating as well as potential drug targets to modulate the channel's function. In addition, we explore the sensitivity of VDAC structure to variations in the membrane environment, comparing DMPC-only, DMPC with cholesterol, and near-native lipid compositions, and use magic-angle spinning NMR spectroscopy to locate cholesterol on the outside of the β-barrel. We find that the VDAC protein structure remains unchanged in different membrane compositions, including conditions with cholesterol. [ABSTRACT FROM AUTHOR]

A contribution of α‐Synuclein (α‐Syn) to etiology of Parkinson´s disease (PD) and Dementia with Lewy bodies (DLB) is currently undisputed, while the impact of the closely related β‐Synuclein (β‐Syn) on these disorders remains enigmatic. β‐Syn has long been considered to be an attenuator of the neurotoxic effects of α‐Syn, but in a rodent model of PD β‐Syn induced robust neurodegeneration in dopaminergic neurons of the substantia nigra. Given that dopaminergic nigral neurons are selectively vulnerable to neurodegeneration in PD, we now investigated if dopamine can promote the neurodegenerative potential of β‐Syn. We show that in cultured rodent and human neurons a dopaminergic neurotransmitter phenotype substantially enhanced β‐Syn‐induced neurodegeneration, irrespective if dopamine is synthesized within neurons or up‐taken from extracellular space. Nuclear magnetic resonance interaction and thioflavin‐T incorporation studies demonstrated that dopamine and its oxidized metabolites 3,4‐dihydroxyphenylacetaldehyde (DOPAL) and dopaminochrome (DCH) directly interact with β‐Syn, thereby enabling structural and functional modifications. Interaction of DCH with β‐Syn inhibits its aggregation, which might result in increased levels of neurotoxic oligomeric β‐Syn. Since protection of outer mitochondrial membrane integrity prevented the additive neurodegenerative effect of dopamine and β‐Syn, such oligomers might act at a mitochondrial level similar to what is suggested for α‐Syn. In conclusion, our results suggest that β‐Syn can play a significant pathophysiological role in etiology of PD through its interaction with dopamine metabolites and thus should be re‐considered as a disease‐relevant factor, at least for those symptoms of PD that depend on degeneration of nigral dopaminergic neurons. [ABSTRACT FROM AUTHOR]

Anisotropic NMR has gained increasing popularity to determine the structure and specifically the configuration of small, flexible, non‐crystallizable molecules. However, it suffers from the necessity to dissolve the analyte in special media such as liquid crystals or polymer gels. Generally, small degrees of alignment are also caused by an anisotropic magnetic susceptibility of the molecule, for example, induced by aromatic moieties. For this mechanism, the alignment can be predicted via density functional theory. Here we show that both residual dipolar couplings and residual chemical shift anisotropies can be acquired from natural products without special sample preparation using magnetically induced alignment. On the two examples of the novel natural product gymnochrome G and the alkaloid strychnine, these data, together with the predicted alignment, yield the correct configuration with high certainty. [ABSTRACT FROM AUTHOR]

Anisotropic NMR has gained increasing popularity to determine the structure and specifically the configuration of small, flexible, non‐crystallizable molecules. However, it suffers from the necessity to dissolve the analyte in special media such as liquid crystals or polymer gels. Generally, small degrees of alignment are also caused by an anisotropic magnetic susceptibility of the molecule, for example, induced by aromatic moieties. For this mechanism, the alignment can be predicted via density functional theory. Here we show that both residual dipolar couplings and residual chemical shift anisotropies can be acquired from natural products without special sample preparation using magnetically induced alignment. On the two examples of the novel natural product gymnochrome G and the alkaloid strychnine, these data, together with the predicted alignment, yield the correct configuration with high certainty. [ABSTRACT FROM AUTHOR]

Parkinson's disease (PD) and Alzheimer's disease (AD) are common neurodegenerative disorders of the elderly and, therefore, affect a growing number of patients worldwide. Both diseases share, as a common hallmark, the accumulation of characteristic protein aggregates, known as Lewy bodies (LB) in PD, and neurofibrillary tangles in AD. LBs are primarily composed of misfolded α-synuclein (aSyn), and neurofibrillary tangles are primarily composed of tau protein. Importantly, upon pathological evaluation, most AD and PD/Lewy body dementia cases exhibit mixed pathology, with the co-occurrence of both LB and neurofibrillary tangles, among other protein inclusions. Recent studies suggest that both aSyn and tau pathology can spread and propagate through neuronal connections. Therefore, it is important to investigate the mechanisms underlying aggregation and propagation of these proteins for the development of novel therapeutic strategies. Here, we assessed the effects of different pharmacological interventions on the aggregation and internalization of tau and aSyn. We found that anle138b and fulvic acid decrease aSyn and tau aggregation, that epigallocatechin gallate decreases aSyn aggregation, and that dynasore reduces tau internalization. Establishing the effects of small molecules with different chemical properties on the aggregation and spreading of aSyn and tau will be important for the development of future therapeutic interventions. [ABSTRACT FROM AUTHOR]

Antibody-mediated immune responses rely on antigen recognition by the B cell antigen receptor (BCR) and the proper engagement of its intracellular signal effector proteins. Src homology (SH) 2 domain-containing leukocyte protein of 65 kDa (SLP65) is the key scaffold protein mediating BCR signaling. In resting B cells, SLP65 colocalizes with Cbl-interacting protein of 85 kDa (CIN85) in cytoplasmic granules whose formation is not fully understood. Here we show that effective B cell activation requires tripartite phase separation of SLP65, CIN85, and lipid vesicles into droplets via vesicle binding of SLP65 and promiscuous interactions between nine SH3 domains of the trimeric CIN85 and the proline-rich motifs (PRMs) of SLP65. Vesicles are clustered and the dynamical structure of SLP65 persists in the droplet phase in vitro. Our results demonstrate that phase separation driven by concerted transient interactions between scaffold proteins and vesicles is a cellular mechanism to concentrate and organize signal transducers. Antibody-mediated immune responses rely on antigen recognition by the B cell antigen receptor (BCR) and SLP65 is a key scaffold protein mediating BCR signaling. Here authors show that effective B cell activation requires tripartite phase separation of SLP65, CIN85, and lipid vesicles. [ABSTRACT FROM AUTHOR]

Nuclear magnetic resonance (NMR) has the unique advantage of elucidating the structure and dynamics of biomolecules in solution at physiological temperatures, where they are in constant movement on timescales from picoseconds to milliseconds. Such motions have been shown to be critical for enzyme catalysis, allosteric regulation, and molecular recognition. With NMR being particularly sensitive to these timescales, detailed information about the kinetics can be acquired. However, nearly all methods of NMR-based biomolecular structure determination neglect kinetics, which introduces a large approximation to the underlying physics, limiting both structural resolution and the ability to accurately determine molecular flexibility. Here we present the Kinetic Ensemble approach that uses a hierarchy of interconversion rates between a set of ensemble members to rigorously calculate Nuclear Overhauser Effect (NOE) intensities. It can be used to simultaneously refine both temporal and structural coordinates. By generalizing ideas from the extended model free approach, the method can analyze the amplitudes and kinetics of motions anywhere along the backbone or side chains. Furthermore, analysis of a large set of crystal structures suggests that NOE data contains a surprising amount of high-resolution information that is better modeled using our approach. The Kinetic Ensemble approach provides the means to unify numerous types of experiments under a single quantitative framework and more fully characterize and exploit kinetically distinct protein states. While we apply the approach here to the protein ubiquitin and cross validate it with previously derived datasets, the approach can be applied to any protein for which NOE data is available. [ABSTRACT FROM AUTHOR]

Type 2 diabetes (T2DM) is associated with aggregation of the human islet amyloid polypeptide (hIAPP) into cytotoxic amyloid species. Here we tested the effect of a diphenylpyrazole (DPP)-derived small molecule inhibitor, anle145c, on cytotoxicity and on aggregation properties of hIAPP. We demonstrate that incubation of hIAPP with the inhibitor yields ~10 nm-sized non-toxic oligomers, independent of the initial aggregation state of hIAPP. This suggests that anle145c has a special mode of action in which anle145c-stabilized oligomers act as a thermodynamic sink for the preferred aggregation state of hIAPP and anle145c. We also demonstrate that the inhibitor acts in a very efficient manner, with sub-stoichiometric concentrations of anle145c being sufficient to (i) inhibit hIAPP-induced death of INS-1E cells, (ii) prevent hIAPP fibril formation in solution, and (iii) convert preformed hIAPP fibrils into non-toxic oligomers. Together, these results indicate that anle145c is a promising candidate for inhibition of amyloid formation in T2DM. [ABSTRACT FROM AUTHOR]

The discovery of aggregation inhibitors and the elucidation of their mechanism of action are key in the quest to mitigate the toxic consequences of amyloid formation. We have previously characterized the antiamyloidogenic mechanism of action of sodium phtalocyanine tetrasulfonate ([Na4(H2PcTS)]) on α-Synuclein (αS), demonstrating that specific aromatic interactions are fundamental for the inhibition of amyloid assembly. Here we studied the influence that metal preferential affinity and peripheral substituents may have on the activity of tetrapyrrolic compounds on αS aggregation. For the first time, our laboratory has extended the studies in the field of the bioinorganic chemistry and biophysics to cellular biology, using a well-established cell-based model to study αS aggregation. The interaction scenario described in our work revealed that both N- and C-terminal regions of αS represent binding interfaces for the studied compounds, a behavior that is mainly driven by the presence of negatively or positively charged substituents located at the periphery of the macrocycle. Binding modes of the tetrapyrrole ligands to αS are determined by the planarity and hydrophobicity of the aromatic ring system in the tetrapyrrolic molecule and/or the preferential affinity of the metal ion conjugated at the center of the macrocyclic ring. The different capability of phthalocyanines and meso-tetra (N-methyl-4-pyridyl) porphine tetrachloride ([H2PrTPCl4]) to modulate αS aggregation in vitro was reproduced in cell-based models of αS aggregation, demonstrating unequivocally that the modulation exerted by these compounds on amyloid assembly is a direct consequence of their interaction with the target protein. [ABSTRACT FROM AUTHOR]

We present an NMR method based on natural abundance 17O relaxation of water to determine effective viscosity in biological aqueous samples. The method accurately captures viscosity of dilute and crowded protein solutions and offers a fairly simple way to quantify the internal fluidity of biological condensates formed through phase separation. [ABSTRACT FROM AUTHOR]

Parkinson's disease (PD) is characterized by the presence of α-synuclein aggregates known as Lewy bodies and Lewy neurites, whose formation is linked to disease development. The causal relation between α-synuclein aggregates and PD is not well understood. We generated a new transgenic mouse line (MI2) expressing human, aggregation-prone truncated 1–120 α-synuclein under the control of the tyrosine hydroxylase promoter. MI2 mice exhibit progressive aggregation of α-synuclein in dopaminergic neurons of the substantia nigra pars compacta and their striatal terminals. This is associated with a progressive reduction of striatal dopamine release, reduced striatal innervation and significant nigral dopaminergic nerve cell death starting from 6 and 12 months of age, respectively. In the MI2 mice, alterations in gait impairment can be detected by the DigiGait test from 9 months of age, while gross motor deficit was detected by rotarod test at 20 months of age when 50% of dopaminergic neurons in the substantia nigra pars compacta are lost. These changes were associated with an increase in the number and density of 20–500 nm α-synuclein species as shown by dSTORM. Treatment with the oligomer modulator anle138b, from 9 to 12 months of age, restored striatal dopamine release, prevented dopaminergic cell death and gait impairment. These effects were associated with a reduction of the inner density of large α-synuclein aggregates and an increase in dispersed small α-synuclein species as revealed by dSTORM. The MI2 mouse model recapitulates the progressive dopaminergic deficit observed in PD, showing that early synaptic dysfunction is associated to fine behavioral motor alterations, precedes dopaminergic axonal loss and neuronal death that become associated with a more consistent motor deficit upon reaching a certain threshold. Our data also provide new mechanistic insight for the effect of anle138b's function in vivo supporting that targeting α-synuclein aggregation is a promising therapeutic approach for PD. [ABSTRACT FROM AUTHOR]

NMR offers many possibilities in chemical analysis, structural investigations, and medical diagnostics. Although it is broadly used, one of NMR spectroscopies main drawbacks is low sensitivity. Hyperpolarization techniques enhance NMR signals by more than four orders of magnitude allowing the design of new contrast agents. Parahydrogen induced polarization that utilizes the para‐hydrogen's singlet state to create enhanced signals is of particular interest since it allows to produce molecular imaging agents within seconds. Herein, we present a strategy for signal enhancement of the carbonyl 13C in amino acids by using parahydrogen, as demonstrated for glycine and alanine. Importantly, the hyperpolarization step is carried out in water and chemically unmodified canonical amino acids are obtained. Our approach thus offers a high degree of biocompatibility, which is crucial for further application. The rapid sample hyperpolarization (within seconds) may enable the continuous production of biologically useful probes, such as metabolic contrast agents or probes for structural biology. [ABSTRACT FROM AUTHOR]

This paper sheds light on the possibilities and perspectives of linking digital editions of Medieval German texts to each other and to other digital resources. Furthermore, it discusses some of the internal and technical conditions necessary to render this linkage meaningful, like lemmatisation, part-of-speech-tagging, and using standardised mark-up languages. Finally, the sustainability and reusability of digital editions are considered. While in the past, editions of medieval texts were conceived as rather isolated scholarly works of individual editors, nowadays the collaboration and cooperation of greater working groups is essential in editing projects. Due to the complexity of editions consisting of multiple textual layers, e. g. apparatus entries, annotations, or facsimiles, the requirements for future digital editions have risen. The first approach to respond to these demands is to link the various textual layers to each other, enabling the users to navigate between these layers in a sensible way. The second step is to link the edited text to other resources, such as online dictionaries or other editions, allowing complex research networks to be created. These goals are achieved by lemmatising and other tagging methods, ensuring the information being mapped to a normalised and idealised frame of reference. Common standards like Unicode or the TEI guidelines are of great importance for such purposes, as they assure the interchange and re-use of scientific data, as well as their sustainability. [ABSTRACT FROM AUTHOR]

Background: MSA is a fatal neurodegenerative disease characterized by autonomic failure and severe motor impairment. Its main pathological hallmark is the accumulation of α-synuclein in oligodendrocytes, leading to glial and neuronal dysfunction and neurodegeneration. These features are recapitulated in the PLP-hαSyn mouse model expressing human α-synuclein in oligodendrocytes. At present, there is no effective disease-modifying therapy. Previous experiments have shown that the aggregation inhibitor, anle138b, reduces neurodegeneration and behavioral deficits in mouse models of other proteinopathies.Objectives: To test the therapeutic potential of anle138b in a mouse model of MSA.Methods: Two-month-old PLP-hαSyn mice were fed over a period of 4 months with pellets containing anle138b at two different doses (0.6 and 2 g/kg) and compared to healthy controls and PLP-hαSyn mice fed with placebo pellets. At the end of the treatment, behavioral and histological analyses were performed.Results: We observed a reversal of motor function to healthy control levels when PLP-hαSyn mice were treated with both doses of anle138b. Histological and molecular analyses showed a significant reduction in α-synuclein oligomers and glial cytoplasmic inclusions in animals fed with anle138b compared to nontreated mice. These animals also present preservation of dopaminergic neurons and reduction in microglial activation in SN correlating with the α-synuclein reduction observed.Conclusions: Anle138b reduces α-synuclein accumulation in PLP-hαSyn mice, leading to neuroprotection, reduction of microglial activation, and preservation of motor function supporting the use of anle138b in a future clinical trial for MSA. © 2018 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society. [ABSTRACT FROM AUTHOR]

Mechanical compression of polymer gels provides a simple way for the measurement of residual chemical shift anisotropies, which then can be employed, on its own, or in combination with residual dipolar couplings, for structural elucidation purposes. Residual chemical shift anisotropies measured using compression devices needed a posteriori correction to account for the increase of the polymer to solvent ratio inside the swollen gel. This correction has been cast before in terms of a single‐free parameter which, as shown here, can be simultaneously optimized along with the components of the alignment tensor while still retaining discriminating power of the different relative configurations as illustrated in the stereochemical analysis of α‐santonin and 10‐epi‐8‐deoxycumambrin B. [ABSTRACT FROM AUTHOR]

Molecular dynamics play a significant role in how molecules perform their function. A critical method that provides information on dynamics, at the atomic level, is NMR-based relaxation dispersion (RD) experiments. RD experiments have been utilized for understanding multiple biological processes occurring at micro-to-millisecond time, such as enzyme catalysis, molecular recognition, ligand binding and protein folding. Here, we applied the recently developed high-power RD concept to the Carr–Purcell–Meiboom–Gill sequence (extreme CPMG; E-CPMG) for the simultaneous detection of fast and slow dynamics. Using a fast folding protein, gpW, we have shown that previously inaccessible kinetics can be accessed with the improved precision and efficiency of the measurement by using this experiment. [ABSTRACT FROM AUTHOR]

Understanding the dissociation of molecules is the basis to modulate interactions of biomedical interest. Optimizing drugs for dissociation rates is found to be important for their efficacy, selectivity, and safety. Here, we show an application of the high-power relaxation dispersion (RD) method to the determination of the dissociation rates of weak binding ligands from receptors. The experiment probes proton RD on the ligand and, therefore, avoids the need for any isotopic labeling. The large ligand excess eases the detection significantly. Importantly, the use of large spin-lock fields allows the detection of faster dissociation rates than other relaxation approaches. Moreover, this experimental approach allows to access directly the off-rate of the binding process without the need for analyzing a series of samples with increasing ligand saturation. The validity of the method is shown with small molecule interactions using two macromolecules, bovine serum albumin and tubulin heterodimers. [ABSTRACT FROM AUTHOR]

Recent epidemiological and clinical studies have reported a significantly increased risk for melanoma in people with Parkinson's disease. Because no evidence could be obtained that genetic factors are the reason for the association between these two diseases, we hypothesized that of the three major Parkinson's disease-related proteins--α-synuclein, LRRK2, and Parkin--α-synuclein might be a major link. Our data, presented here, demonstrate that α-synuclein promotes the survival of primary and metastatic melanoma cells, which is the exact opposite of the effect that α-synuclein has on dopaminergic neurons, where its accumulation causes neuronal dysfunction and death. Because this detrimental effect of α-synuclein on neurons can be rescued by the small molecule anle138b, we explored its effect on melanoma cells. We found that treatment with anle138b leads to massive melanoma cell death due to a major dysregulation of autophagy, suggesting that α-synuclein is highly beneficial to advanced melanoma because it ensures that autophagy is maintained at a homeostatic level that promotes and ensures the cell's survival. [ABSTRACT FROM AUTHOR]