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Humins, (side-)products of the acid-catalysed dehydration of carbohydrates, will be produced in substantial quantities with the development of industrial biorefining processes. Most structural knowledge about such humins is based on synthetic model humins prepared at lab-scale from typical carbohydrate(-derived) compounds. Here, we report the first extensive characterisation study of an industrial humin. The soluble humin was generated from pilot plant-scale methanolic cyclodehydration of D-fructose to 5-methoxymethyl-2-furfural (MMF), as part of the Avantium YXY® process to produce FDCA. Purification of the industrial humin followed by fractionation allowed isolation of a water-insoluble, high molecular weight fraction (WIPIH) and a water-soluble, low-to-middle molecular weight soluble fraction (WES). Characterisation by elemental analysis, thermogravimetry, IR and NMR spectroscopy and size exclusion chromatography provided a detailed picture of the humin structure in both fractions. Aided by a comprehensive NMR spectral library of furanic model compounds, we identified the main furanic building blocks and inter-unit linkages and propose a structure for this industrial humin sample. The WIPIH and WES fractions were found to be composed of furanic rings interconnected by short aliphatic chains containing a wide range of functionalities including alcohols, ethers, carboxylic acids, esters, aldehydes and ketones. The low level of crosslinking and high functional group content of the industrial humin differ from the more extensively studied, (highly over-)condensed synthetic model humins, towards which they can be considered intermediates. The structural and compositional insights into the nature of an actual industrial humin open up a broad spectrum of valorisation opportunities. [ABSTRACT FROM AUTHOR]

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

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

Herein, we investigate a novel set of polarizing agents—mixed‐valence compounds—by theoretical and experimental methods and demonstrate their performance in high‐field dynamic nuclear polarization (DNP) NMR experiments in the solid state. Mixed‐valence compounds constitute a group of molecules in which molecular mobility persists even in solids. Consequently, such polarizing agents can be used to perform Overhauser‐DNP experiments in the solid state, with favorable conditions for dynamic nuclear polarization formation at ultra‐high magnetic fields. [ABSTRACT FROM AUTHOR]

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

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

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

Abstract: After a prolonged effort over many years, the route for the formation of a direct carbon−carbon (C−C) bond during the methanol‐to‐hydrocarbon (MTH) process has very recently been unveiled. However, the relevance of the “direct mechanism”‐derived molecules (that is, methyl acetate) during MTH, and subsequent transformation routes to the conventional hydrocarbon pool (HCP) species, are yet to be established. This important piece of the MTH chemistry puzzle is not only essential from a fundamental perspective, but is also important to maximize catalytic performance. The MTH process was probed over a commercially relevant H‐SAPO‐34 catalyst, using a combination of advanced solid‐state NMR spectroscopy and operando UV/Vis diffuse reflectance spectroscopy coupled to an on‐line mass spectrometer. Spectroscopic evidence is provided for the formation of (olefinic and aromatic) HCP species, which are indeed derived exclusively from the direct C−C bond‐containing acetyl group of methyl acetate. New mechanistic insights have been obtained from the MTH process, including the identification of hydrocarbon‐based co‐catalytic organic reaction centers. [ABSTRACT FROM AUTHOR]

Abstract: Chromatin function depends on a dense network of interactions between nucleosomes and a wide range of proteins. A detailed description of these protein–nucleosome interactions is required to reach a full molecular understanding of chromatin function in both genetics and epigenetics. Herein, we show that the structure, dynamics, and interactions of nucleosomes can be interrogated in a residue‐specific manner by using state‐of‐the‐art solid‐state NMR spectroscopy. Using sedimented nucleosomes, high‐resolution spectra were obtained for both flexible histone tails and the non‐mobile histone core. Through co‐sedimentation of a nucleosome‐binding peptide, we demonstrate that protein‐binding sites on the nucleosome surface can be determined. We believe that this approach holds great promise as it is generally applicable, extendable to include the structure and dynamics of the bound proteins, and scalable to interactions of proteins with higher‐order chromatin structures, including isolated and cellular chromatin. [ABSTRACT FROM AUTHOR]

Methanol-to-olefin (MTO) catalysis is a very active field of research because there is a wide variety of sometimes conflicting mechanistic proposals. An example is the ongoing discussion on the initial C−C bond formation from methanol during the induction period of the MTO process. By employing a combination of solid-state NMR spectroscopy with UV/Vis diffuse reflectance spectroscopy and mass spectrometry on an active H-SAPO-34 catalyst, we provide spectroscopic evidence for the formation of surface acetate and methyl acetate, as well as dimethoxymethane during the MTO process. As a consequence, new insights in the formation of the first C−C bond are provided, suggesting a direct mechanism may be operative, at least in the early stages of the MTO reaction. [ABSTRACT FROM AUTHOR]

1H detection can significantly improve solid-state NMR spectral sensitivity and thereby allows studying more complex proteins. However, the common prerequisite for 1H detection is the introduction of exchangeable protons in otherwise deuterated proteins, which has thus far significantly hampered studies of partly water-inaccessible proteins, such as membrane proteins. Herein, we present an approach that enables high-resolution 1H-detected solid-state NMR (ssNMR) studies of water-inaccessible proteins, and that even works in highly complex environments such as cellular surfaces. In particular, the method was applied to study the K+ channel KcsA in liposomes and in situ in native bacterial cell membranes. We used our data for a dynamic analysis, and we show that the selectivity filter, which is responsible for ion conduction and highly conserved in K+ channels, undergoes pronounced molecular motion. We expect this approach to open new avenues for biomolecular ssNMR. [ABSTRACT FROM AUTHOR]

1H-detection can greatly improve spectral sensitivity in biological solid-state NMR (ssNMR), thus allowing the study of larger and more complex proteins. However, the general requirement to perdeuterate proteins critically curtails the potential of 1H-detection by the loss of aliphatic side-chain protons, which are important probes for protein structure and function. Introduced herein is a labelling scheme for 1H-detected ssNMR, and it gives high quality spectra for both side-chain and backbone protons, and allows quantitative assignments and aids in probing interresidual contacts. Excellent 1H resolution in membrane proteins is obtained, the topology and dynamics of an ion channel were studied. This labelling scheme will open new avenues for the study of challenging proteins by ssNMR. [ABSTRACT FROM AUTHOR]

Cross-effect (CE) dynamic nuclear polarization (DNP) is a rapidly developing technique that enhances the signal intensities in magic-angle spinning (MAS) NMR spectra. We report CE DNP experiments at 211, 600, and 800 MHz using a new series of biradical polarizing agents referred to as TEMTriPols, in which a nitroxide (TEMPO) and a trityl radical are chemically tethered. The TEMTriPol molecule with the optimal performance yields a record 1H NMR signal enhancement of 65 at 800 MHz at a concentration of 10 m M in a glycerol/water solvent matrix. The CE DNP enhancement for the TEMTriPol biradicals does not decrease as the magnetic field is increased in the manner usually observed for bis-nitroxides. Instead, the relatively strong exchange interaction between the trityl and nitroxide moieties determines the magnetic field at which the optimum enhancement is observed. [ABSTRACT FROM AUTHOR]

Dynamic nuclear polarization (DNP) has been shown to greatly enhance spectroscopic sensitivity, creating novel opportunities for NMR studies on complex and large molecular assemblies in life and material sciences. In such applications, however, site-specificity and spectroscopic resolution become critical factors that are usually difficult to control by current DNP-based approaches. We have examined in detail the effect of directly attaching mono- or biradicals to induce local paramagnetic relaxation effects and, at the same time, to produce sizable DNP enhancements. Using a membrane-embedded ion channel as an example, we varied the degree of paramagnetic labeling and the location of the DNP probes. Our results show that the creation of local spin clusters can generate sizable DNP enhancements while preserving the intrinsic benefits of paramagnetic relaxation enhancement (PRE)-based NMR approaches. DNP using chemical labeling may hence provide an attractive route to introduce molecular specificity into DNP studies in life science applications and beyond. [ABSTRACT FROM AUTHOR]

Humin by-products are formed during the acid-catalyzed dehydration of carbohydrates to bio-based platform molecules, such as hydroxymethylfurfural and levulinic acid. The molecular structure of these humins has not yet been unequivocally established. 1D 13C solid-state NMR data reported have, for example, provided considerable insight, but do not allow for the unambiguous assignment of key structural motifs. Complementary (2D) techniques are needed to gain additional insight into the molecular structure of humins. Here, the preparation of 13C-enriched humins is reported, together with the reactive solubilization of these labeled humins and their characterization with complementary 1D and 2D solid-state NMR techniques. 1D cross polarization (CP) and direct excitation (DE) 13C solid-state NMR spectra, 2D 13C-detected double-quantum single-quantum (DQSQ) as well as 2D 1H-detected heteronuclear correlation (HETCOR) were recorded with different excitation schemes. These experiments unambiguously established that the original humins have a furan-rich structure with aliphatic linkers and allowed for a refinement of the molecular structure proposed previously. Solid-state NMR data of alkali-treated 13C-labeled humins showed that an arene-rich structure is formed at the expense of the furanic network during alkaline pretreatment. [ABSTRACT FROM AUTHOR]

Sensitivity is the key: Dynamic nuclear polarization NMR spectroscopy provides structural information on liposomal vaccines targeting Alzheimer's disease. DMPC/DMPG/Cholesterol mainly stabilizes extended structures of the lipid‐anchored peptide, while in DMTAP/Cholesterol liposomes the peptide adopts a multitude of conformations including random‐coil and extended structures. [ABSTRACT FROM AUTHOR]

Supramolecular chemistry provides structural and conformational information about complexes formed from multiple molecules. While the molecule is held together by strong intramolecular contacts like covalent bonds, supramolecular structures can be further stabilized by weaker or transient intermolecular interactions. These interactions can confer a great diversity and sensitivity to exogenous factors like temperature, pressure, or ionic strength to multimolecular arrangements. [ABSTRACT FROM AUTHOR]

Members of the histone-like nucleoid structuring protein ( H- NS) family play roles both as architectural proteins and as modulators of gene expression in Gram-negative bacteria. The H- NS protein participates in modulatory processes that respond to environmental changes in osmolarity, pH, or temperature. H- NS oligomerization is essential for its activity. Structural models of different truncated forms are available. However, high-resolution structural details of full-length H- NS and its DNA-bound state have largely remained elusive. We report on progress in characterizing the biologically active H- NS oligomers with solid-state NMR. We compared uniformly (13 C,15 N)-labeled ss NMR preparations of the isolated N-terminal region ( H- NS 1-47) and full-length H- NS ( H- NS 1-137). In both cases, we obtained ss NMR spectra of good quality and characteristic of well-folded proteins. Analysis of the results of 2 D and 3 D 13 C-13 C and 15 N-13 C correlation experiments conducted at high magnetic field led to assignments of residues located in different topological regions of the free full-length H- NS. These findings confirm that the structure of the N-terminal dimerization domain is conserved in the oligomeric full-length protein. Small changes in the dimerization interface suggested by localized chemical shift variations between solution and solid-state spectra may be relevant for DNA recoginition. Database Structural data are available in the BioMagResBank database ( BMRB; http://www.bmrb.wisc.edu) under accession number 18814. Structured digital abstract H-NS and H-NS bind by nuclear magnetic resonance (View interaction) [ABSTRACT FROM AUTHOR]

High-resolution solid-state NMR spectroscopy can provide structural information of proteins that cannot be studied by X-ray crystallography or solution NMR spectroscopy. Here we demonstrate that it is possible to determine a protein structure by solid-state NMR to a resolution comparable to that by solution NMR. Using an iterative assignment and structure calculation protocol, a large number of distance restraints was extracted from 1H/1H mixing experiments recorded on a single uniformly labeled sample under magic angle spinning conditions. The calculated structure has a coordinate precision of 0.6 Å and 1.3 Å for the backbone and side chain heavy atoms, respectively, and deviates from the structure observed in solution. The approach is expected to be applicable to larger systems enabling the determination of high-resolution structures of amyloid or membrane proteins. [ABSTRACT FROM AUTHOR]

The 140-residue protein α-synuclein (AS) is able to form amyloid fibrils and as such is the main component of protein inclusions involved in Parkinson's disease. We have investigated the structure and dynamics of full-length AS fibrils by high-resolution solid-state NMR spectroscopy. Homonuclear and heteronuclear 2D and 3D spectra of fibrils grown from uniformly 13C/15N-labeled AS and AS reverse-labeled for two of the most abundant amino acids, K and V. were analyzed. 13C and 15N signals exhibited linewidths of <0.7 ppm. Sequential assignments were obtained for 48 residues in the hydrophobic core region. We identified two different types of fibrils displaying chemical-shift differences of up to 13 ppm in the 15N dimension and up to 5 ppm for backbone and side-chain 13C chemical shifts. EM studies suggested that molecular structure is correlated with fibril morphology. Investigation of the secondary structure revealed that most amino acids of the core region belong to β-strands with similar torsion angles in both conformations. Selection of regions with different mobility indicated the existence of monomers in the sample and allowed the identification of mobile segments of the protein within the fibril in the presence of monomeric protein. At least 35 C-terminal residues were mobile and lacked a defined secondary structure, whereas the N terminus was rigid starting from residue 22. Our findings agree well with the overall picture obtained with other methods and provide insight into the amyloid fibril structure and dynamics with residue-specific resolution. [ABSTRACT FROM AUTHOR]

Rücktitelbild: DNP-Supported Solid-State NMR Spectroscopy of Proteins Inside Mammalian Cells (Angew. Keywords: Dynamische Kernpolarisation; Festkörper-NMR-Spektroskopie; NMR-Spektroskopie in Zellen; Protein-Protein-Wechselwirkungen; Ubiquitinierung B Durch dynamische Kernpolarisation (DNP) b verstärkte Festkörper-NMR-Spektroskopie (ssNMR) ermöglicht strukturelle Studien an isotopenmarkierten Proteinen auf atomarem Niveau bei physiologisch relevanten Konzentrationen in Säugerzellen, unabhängig von der Taumelgeschwindigkeit des jeweiligen Proteins. [Extracted from the article]

Review: 38 refs. [ABSTRACT FROM AUTHOR]

[Display omitted] • Structure and function of the osm otically inducible protein Y (OsmY) in Escherichia coli. • OsmY interacts with AquaporinZ under high ionic strength conditions, suggesting a connection with the response of Escherichia coli to osmotic stress. • Insights into bacterial survival strategies in osmotically diverse environments. • OsmY's function is dependent on its primary Bacterial OsmY and Nodulation.domain BON1. • OsmY is a key player in facilitating cell volume recovery and adaptation to severe osmotic stress in Escherichia coli and likely other Gram-negative bacteria. 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. [ABSTRACT FROM AUTHOR]

Abstract Significant progress has been made in obtaining structural insight into the assembly of the β-barrel assembly machinery complex (BAM). These crystallography and electron microscopy studies used detergent as a membrane mimetic and revealed structural variations in the central domain, BamA, as well as in the lipoprotein BamC. We have used cellular solid-state NMR spectroscopy to examine the entire BamABCDE complex in native outer membranes and obtained data on the BamCDE subcomplex in outer membranes, in addition to synthetic bilayers. To reduce spectral crowding, we utilized proton-detected experiments and employed amino-acid specific isotope-labelling in (13C, 13C) correlation experiments. Taken together, the results provide insight into the overall fold and assembly of the BAM complex in native membranes, in particular regarding the structural flexibility of BamC in the absence of the core unit BamA. [ABSTRACT FROM AUTHOR]

Recovery in K+ channels, that is, the transition from the inactivated nonconductive selectivity filter conformation toward the conductive conformation, occurs on a time scale of the order of seconds, which is astonishingly long, given that the structural differences among the filter conformations are faint (<1 Å). Computational studies and electrophysiological measurements suggested that buried water molecules bound behind the selectivity filter are at the origin of the slowness of recovery in K+ channels. Using a combination of solid-state NMR spectroscopy (ssNMR) and long molecular dynamics simulations, we sketch a high-resolution map of the spatial and temporal distribution of water behind the selectivity filter of a membrane-embedded K+ channel in two different gating modes. Our study demonstrates that buried water molecules with long residence times are spread all along the rear of the inactivated filter, which explains the recovery kinetics. In contrast, the same region of the structure appears to be dewetted when the selectivity filter is in the conductive state. Using proton-detected ssNMR on fully protonated channels, we demonstrate the presence of a pathway that allows for the interchange of buried and bulk water, as required for a functional influence of buried water on recovery and slow inactivation. Furthermore, we provide direct experimental evidence for the presence of additional ordered water molecules that surround the filter and that are modulated by the channel's gating mode. [ABSTRACT FROM AUTHOR]

Thermosensitiveamphiphilic block copolymers self-assemble intomicelles above their lower critical solution temperature in water,however, the micelles generally display mediocre physical stability.To stabilize such micelles and increase their loading capacity forchemotherapeutic drugs, block copolymers with novel aromatic monomerswere synthesized by free radical polymerization of N-(2-benzoyloxypropyl methacrylamide (HPMAm-Bz) or the correspondingnaphthoyl analogue (HPMAm-Nt), with N-(2-hydroxypropyl)methacrylamide monolactate, using a polyethylene glycol based macroinitiator.The critical micelle temperatures and critical micelle concentrationsdecreased with increasing the HPMAm-Bz/Nt content. The micelles of30–50 nm were prepared by heating the polymer aqueous solutionsfrom 0 to 50 °C and were colloidally stable for at least 48 hat pH 7.4 and 37 °C. Paclitaxel and docetaxel encapsulation wasperformed by mixing drug solutions in ethanol with polymer aqueoussolutions and heating from 0 to 50 °C. The micelles had a drugloading capacity up to 34 wt % for docetaxel, which is among the highestloadings reported for polymeric micelles, with loaded micelle sizesranging from 60 to 80 nm. The micelles without aromatic groups almostcompletely released loaded paclitaxel in 10 days, whereas the HPMAm-Bz/Ntcontaining micelles released 50% of the paclitaxel at the same time,which showed a better retention for the drug of the latter micelles. 1H solid-state NMR spectroscopy data are compatible with π–πstacking between aromatic groups. The empty micelles demonstratedgood cytocompatibility, and paclitaxel-loaded micelles showed highcytotoxicity to tumor cells. In conclusion, the π–πstacking effect introduced by aromatic groups increases the stabilityand loading capacity of polymeric micelles. [ABSTRACT FROM AUTHOR]

One of the hallmarks of Alzheimer’s disease is the self-assembly of the microtubule-associated protein tau into fibers termed “paired helical filaments” (PHFs). However, the structural basis of PHF assembly at atomic detail is largely unknown. Here, we applied solid-state nuclear magnetic resonance (ssNMR) spectroscopy to investigate in vitro assembled PHFs from a truncated three-repeat tau isoform (K19) that represents the core of PHFs. We found that the rigid core of the fibrils is formed by amino acids V306 to S324, only 18 out of 99 residues, and comprises three β-strands connected by two short kinks. The first β-strand is formed by the well-studied hexapeptide motif VQIVYK that is known to self-aggregate in a steric zipper arrangement. Results on mixed [15N:13C]-labeled K19 fibrils show that β-strands are stacked in a parallel, in-register manner. Disulfide bridges formed between C322 residues of different molecules lead to a disturbance of the β-sheet structure, and polymorphism in ssNMR spectra is observed. In particular, residues K321-S324 exhibit two sets of resonances. Experiments on K19 C322A PHFs further confirm the influence of disulfide bond formation on the core structure. Our structural data are supported by H/D exchange NMR measurements on K19 as well as a truncated four-repeat isoform of tau (K18). Site-directed mutagenesis studies show that single-point mutations within the three different β-strands result in a significant loss of PHF aggregation efficiency, highlighting the importance of the β-structure-rich regions for tau aggregation. [ABSTRACT FROM AUTHOR]

Abstract: Protein aggregation via polyglutamine stretches occurs in a number of severe neurodegenerative diseases such as Huntington''s disease. We have investigated fibrillar aggregates of polyglutamine peptides below, at, and above the toxicity limit of around 37 glutamine residues using solid-state NMR and electron microscopy. Experimental data are consistent with a dry fibril core of at least 70–80 Å in width for all constructs. Solid-state NMR dipolar correlation experiments reveal a largely β-strand character of all samples and point to tight interdigitation of hydrogen-bonded glutamine side chains from different sheets. Two approximately equally frequent populations of glutamine residues with distinct sets of chemical shifts are found, consistent with local backbone dihedral angles compensating for β-strand twist or with two distinct sets of side-chain conformations. Peptides comprising 15 glutamine residues are present as single extended β-strands. Data obtained for longer constructs are most compatible with a superpleated arrangement with individual molecules contributing β-strands to more than one sheet and an antiparallel assembly of strands within β-sheets. [Copyright &y& Elsevier]