An evolutionarily conserved mechanism controls reversible amyloids of pyruvate kinase via pH-sensing regions.

Amyloids are known as irreversible aggregates associated with neurodegenerative diseases. However, recent evidence shows that a subset of amyloids can form reversibly and fulfill essential cellular functions. Yet, the molecular mechanisms regulating functional amyloids and distinguishing them from pathological aggregates remain unclear. Here, we investigate the conserved principles of amyloid reversibility by studying the essential metabolic enzyme pyruvate kinase (PK) in yeast and human cells. We demonstrate that yeast PK (Cdc19) and human PK (PKM2) form reversible amyloids through a pH-sensitive amyloid core. Stress-induced cytosolic acidification promotes aggregation via protonation of specific glutamate (yeast) or histidine (human) residues within the amyloid core. Mutations mimicking protonation cause constitutive PK aggregation, while non-protonatable PK mutants remain soluble even upon stress. Physiological PK aggregation is coupled to metabolic rewiring and glycolysis arrest, causing severe growth defects when misregulated. Our work thus identifies an evolutionarily conserved, potentially widespread mechanism regulating functional amyloids during stress.

The assembly platform FimD is required to obtain the most stable quaternary structure of type 1 pili.

Type 1 pili are important virulence factors of uropathogenic Escherichia coli that mediate bacterial attachment to epithelial cells in the urinary tract. The pilus rod is comprised of thousands of copies of the main structural subunit FimA and is assembled in vivo by the assembly platform FimD. Although type 1 pilus rods can self-assemble from FimA in vitro, this reaction is slower and produces structures with lower kinetic stability against denaturants compared to in vivo-assembled rods. Our study reveals that FimD-catalysed in vitro-assembled type 1 pilus rods attain a similar stability as pilus rods assembled in vivo. Employing structural, biophysical and biochemical analyses, we show that in vitro assembly reactions lacking FimD produce pilus rods with structural defects, reducing their stability against dissociation. Overall, our results indicate that FimD is not only required for the catalysis of pilus assembly, but also to control the assembly of the most stable quaternary structure.

Initial Primer Synthesis of a DNA Primase Monitored by Real-Time NMR Spectroscopy.

Primases are crucial enzymes for DNA replication, as they synthesize a short primer required for initiating DNA replication. We herein present time-resolved nuclear magnetic resonance (NMR) spectroscopy in solution and in the solid state to study the initial dinucleotide formation reaction of archaeal pRN1 primase. Our findings show that the helix-bundle domain (HBD) of pRN1 primase prepares the two substrates and then hands them over to the catalytic domain to initiate the reaction. By using nucleotide triphosphate analogues, the reaction is substantially slowed down, allowing us to study the initial dinucleotide formation in real time. We show that the sedimented protein-DNA complex remains active in the solid-state NMR rotor and that time-resolved 31P-detected cross-polarization experiments allow monitoring the kinetics of dinucleotide formation. The kinetics in the sedimented protein sample are comparable to those determined by solution-state NMR. Protein conformational changes during primer synthesis are observed in time-resolved 1H-detected experiments at fast magic-angle spinning frequencies (100 kHz). A significant number of spectral changes cluster in the HBD pointing to the importance of the HBD for positioning the nucleotides and the dinucleotide.

7Li NMR Spectroscopy: A Tool for Determining Dimerization Constants and Averaged Dimerization Constants of the Monomer/Dimer Equilibrium of Hierarchical Helicates.

7Li nuclear magnetic resonance (NMR) spectroscopy is an ideal tool to study hierarchically assembled helicates of the form Li[Li3L6Ti2]. Internally bound and external lithium ions can be well distinguished by solution- or solid-state NMR spectroscopy and dimerization constants of the monomer/dimer equilibrium can be easily determined in solution. Averaged dimerization constants can be estimated in case of statistical mixtures of helicates formed from mixtures of ligands.

Triclinic La7Zn2P11 with P3-, P24-, and P35- units: a combined study by 31P solid-state NMR spectroscopy and single crystal X-ray diffraction.

The ternary polyphosphide La7Zn2P11 was synthesized from the elements by using a salt flux or via a ceramic method in sealed quartz ampoules. The obtained samples were investigated by X-ray powder and single crystal diffraction: own type, P1̄, a = 775.33(13), b = 827.45(13), c = 1502.8(3) pm, α = 82.111(3), ß = 77.034(3), γ = 89.996(3)°, wR2 = 0.1553, 5852 F2 values and 183 variables. This peculiar structure is characterized by the simultaneous presence of three distinct anionic phosphide species, namely P3-, P24-, and P35- units. La7Zn2P11 is an electron precise Zintl phase: (7La3+)21+(2Zn2+)4+(4P3-)12-(2P24-)8-(P35-). The P-P single bond distances range from 219.2 to 223.0 pm. The zinc sites show tetrahedral phosphorus coordination by three P3- and one P24- species. The tetrahedra are condensed to chains via common corners. The P35- units with P-P-P angles of 113.7° have exclusively lanthanum coordination. 31P solid-state NMR was used to probe the phosphorus local environments, connectivities and spatial proximities. The eleven crystallographically distinct phosphorus atoms were assigned with the help of two-dimensional homonuclear dipolar correlation experiments. Even though the application of 2D measurements on such phosphorus-based polyanionic compounds is exceedingly challenging because of the wide dispersion of chemical shifts, the fast irreversible decay of the transverse magnetization, and slow spin-lattice relaxation, a complete assignment is possible using radiofrequency-driven dipolar recoupling (RFDR), J-RESOLVED and total-through-bond correlation with R-sequence (R-TOBSY) techniques.

A solid beta-sheet structure is formed at the surface of FUS droplets during aging.

Phase transitions are important to understand cell dynamics, and the maturation of liquid droplets is relevant to neurodegenerative disorders. We combined NMR and Raman spectroscopies with microscopy to follow, over a period of days to months, droplet maturation of the protein fused in sarcoma (FUS). Our study reveals that the surface of the droplets plays a critical role in this process, while RNA binding prevents it. The maturation kinetics are faster in an agarose-stabilized biphasic sample compared with a monophasic condensed sample, owing to the larger surface-to-volume ratio. In addition, Raman spectroscopy reports structural differences upon maturation between the inside and the surface of droplets, which is comprised of ß-sheet content, as revealed by solid-state NMR. In agreement with these observations, a solid crust-like shell is observed at the surface using microaspiration. Ultimately, matured droplets were converted into fibrils involving the prion-like domain as well as the first RGG motif.

Polar covalent apex-base bonding in borapyramidanes probed by solid-state NMR and DFT calculations.

Pyramidane molecules have attracted chemists for many decades due to their regular shape, high symmetry and their correspondence in the macroscopic world. Recently, experimental access to a number of examples has been reported, in particular the rarely reported square pyramidal bora[4]pyramidanes. To describe the bonding situation of the nonclassical structure of pyramidanes, we present solid-state Nuclear Magnetic Resonance (NMR) as a versatile tool for deciphering such bonding properties for three now accessible bora[4]pyramidane and dibora[5]pyramidane molecules. 11 B solid-state NMR spectra indicate that the apical boron nuclei in these compounds are strongly shielded (around -50 ppm vs. BF3 -Et2 O complex) and possess quadrupolar coupling constants of less than 0.9 MHz pointing to a rather high local symmetry. 13 C-11 B spin-spin coupling constants have been explored as a measure of the bond covalency in the borapyramidanes. While the carbon-boron bond to the -B(C6 F5 )2 substituents of the base serves as an example for a classical covalent 2-center-2-electron (2c-2e) sp2 -carbon-sp2 -boron σ-bond with 1 J(13 C-11 B) coupling constants in the order of 75 Hz, those of the boron(apical)-carbon(basal) bonds in the pyramid are too small to measure. These results suggest that these bonds have a strongly ionic character, which is also supported by quantum-chemical calculations.

An Analysis of Nucleotide-Amyloid Interactions Reveals Selective Binding to Codon-Sized RNA.

Interactions between RNA and proteins are the cornerstone of many important biological processes from transcription and translation to gene regulation, yet little is known about the ancient origin of said interactions. We hypothesized that peptide amyloids played a role in the origin of life and that their repetitive structure lends itself to building interfaces with other polymers through avidity. Here, we report that short RNA with a minimum length of three nucleotides binds in a sequence-dependent manner to peptide amyloids. The 3'-5' linked RNA backbone appears to be well-suited to support these interactions, with the phosphodiester backbone and nucleobases both contributing to the affinity. Sequence-specific RNA-peptide interactions of the kind identified here may provide a path to understanding one of the great mysteries rooted in the origin of life: the origin of the genetic code.

High and fast: NMR protein-proton side-chain assignments at 160 kHz and 1.2 GHz.

The NMR spectra of side-chain protons in proteins provide important information, not only about their structure and dynamics, but also about the mechanisms that regulate interactions between macromolecules. However, in the solid-state, these resonances are particularly difficult to resolve, even in relatively small proteins. We show that magic-angle-spinning (MAS) frequencies of 160 kHz, combined with a high magnetic field of 1200 MHz proton Larmor frequency, significantly improve their spectral resolution. We investigate in detail the gain for MAS frequencies between 110 and 160 kHz MAS for a model sample as well as for the hepatitis B viral capsid assembled from 120 core-protein (Cp) dimers. For both systems, we found a significantly improved spectral resolution of the side-chain region in the 1H-13C 2D spectra. The combination of 160 kHz MAS frequency with a magnetic field of 1200 MHz, allowed us to assign 61% of the aliphatic protons of Cp. The side-chain proton assignment opens up new possibilities for structural studies and further characterization of protein-protein or protein-nucleic acid interactions.

Adaptive Catalysts for the Selective Hydrogenation of Bicyclic Heteroaromatics using Ruthenium Nanoparticles on a CO2 -Responsive Support.

Ruthenium nanoparticles (NPs) immobilized on an amine-functionalized polymer-grafted silica support act as adaptive catalysts for the hydrogenation of bicyclic heteroaromatics. Whereas full hydrogenation of benzofuran and quinoline derivatives is achieved under pure H2 , introducing CO2 into the H2 gas phase leads to an effective shutdown of the arene hydrogenation while preserving the activity for the hydrogenation of the heteroaromatic part. The selectivity switch originates from the generation of ammonium formate species on the surface of the materials by catalytic hydrogenation of CO2 . The CO2 hydrogenation is fully reversible, resulting in a robust and rapid switch between the two states of the catalyst adapting its performance in response to the feed gas composition. A variety of benzofuran and quinoline derivatives were hydrogenated to fully or partially saturated products in high selectivity and yields simply by altering the composition of the feed gas from H2 to H2 /CO2 . The adaptive catalytic system thus provides controlled access to valuable products using a single catalyst rather than two specific and distinct catalysts with static reactivity.

A Simple and Versatile Approach for the Low-Temperature Synthesis of Transition Metal Phosphide Nanoparticles from Metal Chloride Complexes and P(SiMe3 )3.

Metal chloride complexes react with tris(trimethylsilyl)phosphine under mild condition to produce metal phosphide (TMP) nanoparticles (NPs), and chlorotrimethylsilane as a byproduct. The formation of Si-Cl bonds that are stronger than the starting M-Cl bonds acts as a driving force for the reaction. The potential of this strategy is illustrated through the preparation of ruthenium phosphide NPs using [RuCl2 (cymene)] and tris(trimethylsilyl)phosphine at 35 °C. Characterization with a combination of techniques including electron microscopy (EM), X-ray absorption spectroscopy (XAS), and solid-state nuclear magnetic resonance (NMR) spectroscopy, evidences the formation of small (diameter of 1.3 nm) and amorphous NPs with an overall Ru50 P50 composition. Interestingly, these NPs can be easily immobilized on functional support materials, which is of great interest for potential applications in catalysis and electrocatalysis. Mo50 P50 and Co50 P50 NPs can also be synthesized following the same strategy. This approach is simple and versatile and paves the way toward the preparation of a wide range of transition metal phosphide nanoparticles under mild reaction conditions.

Opportunities and Challenges in Applying Solid-State NMR Spectroscopy in Organic Mechanochemistry.

In recent years it is shown that mechanochemical strategies can be beneficial in directed conversions of organic compounds. Finding new reactions proved difficult, and due to the lack of mechanistic understanding of mechanochemical reaction events, respective efforts have mostly remained empirical. Spectroscopic techniques are crucial in shedding light on these questions. In this overview, the opportunities and challenges of solid-state nuclear magnetic resonance (NMR) spectroscopy in the field of organic mechanochemistry are discussed. After a brief discussion of the basics of high-resolution solid-state NMR under magic-angle spinning (MAS) conditions, seven opportunities for solid-state NMR in the field of organic mechanochemistry are presented, ranging from ex situ approaches to structurally elucidated reaction products obtained by milling to the potential and limitations of in situ solid-state NMR approaches. Particular strengths of solid-state NMR, for instance in differentiating polymorphs, in NMR-crystallographic structure-determination protocols, or in detecting weak noncovalent interactions in molecular-recognition events employing proton-detected solid-state NMR experiments at fast MAS frequencies, are discussed.

The effect of methyl group rotation on 1H-1H solid-state NMR spin-diffusion spectra.

Fast magic-angle spinning (MAS) NMR experiments open the way for proton-detected NMR studies and have been explored in the past years for a broad range of materials, comprising biomolecules and pharmaceuticals. Proton-spin diffusion (SD) is a versatile polarization-transfer mechanism and plays an important role in resonance assignment and structure determination. Recently, the occurrence of negative cross peaks in 2D 1H-1H SD-based spectra has been reported and explained with higher-order SD effects, in which the chemical shifts of the involved quadruple of nuclei need to compensate each other. We herein report negative cross peaks in SD-based spectra observed for a variety of small organic molecules involving methyl groups. We combine experimental observations with numerical and analytical simulations to demonstrate that the methyl groups can give rise to coherent (SD) as well as incoherent (Nuclear Overhauser Enhancement, NOE) effects, both in principle manifesting themselves as negative cross peaks in the 2D spectra. Analytical calculations and simulations however show that higher-order coherent contributions dominate the experimentally observed negative peaks in our systems. Methyl groups are prone to the observation of such higher order coherent effects. Due to their low-frequency shifted 1H resonances, the chemical-shift separation relative to for instance aromatic protons in spatial proximity is substantial (>4.7 ppm in the studied examples) preventing any sizeable second-order spin-diffusion processes, which would mask the negative contribution to the peaks.

Fibrils Emerging from Droplets: Molecular Guiding Principles behind Phase Transitions of a Short Peptide-Based Condensate Studied by Solid-State NMR.

Biochemical reactions occurring in highly crowded cellular environments require different means of control to ensure productivity and specificity. Compartmentalization of reagents by liquid-liquid phase separation is one of these means. However, extremely high local protein concentrations of up to 400 mg/ml can result in pathological aggregation into fibrillar amyloid structures, a phenomenon that has been linked to various neurodegenerative diseases. Despite its relevance, the process of liquid-to-solid transition inside condensates is still not well understood at the molecular level. We thus herein use small peptide derivatives that can undergo both liquid-liquid and subsequent liquid-to-solid phase transition as model systems to study both processes. Using solid-state nuclear magnetic resonance (NMR) and transmission electron microscopy (TEM), we compare the structure of condensed states of leucine, tryptophan and phenylalanine containing derivatives, distinguishing between liquid-like condensates, amorphous aggregates and fibrils, respectively. A structural model for the fibrils formed by the phenylalanine derivative was obtained by an NMR-based structure calculation. The fibrils are stabilised by hydrogen bonds and side-chain π-π interactions, which are likely much less pronounced or absent in the liquid and amorphous state. Such noncovalent interactions are equally important for the liquid-to-solid transition of proteins, particularly those related to neurodegenerative diseases.

Rethinking global digital health and AI-for-health innovation challenges.

Digital health technologies can help tackle challenges in global public health. Digital and AI-for-Health Challenges, controlled events whose goal is to generate solutions to a given problem in a defined period of time, are one way of catalysing innovation. This article proposes an expanded investment framework for Global Health AI and digitalhealth Innovation that goes beyond traditional factors such as return on investment. Instead, we propose non monetary and non GDP metrics, such as Disability Adjusted Life Years or achievement of universal health coverage. Furthermore, we suggest a venture building approach around global health, which includes filtering of participants to reduce opportunity cost, close integration of implementation scientists and an incubator for the long-term development of ideas resulting from the challenge. Finally, we emphasize the need to strengthen human capital across a range of areas in local innovation, implementation-science, and in health services.

Region-based Template Matching Prediction for Intra Coding.

Copy prediction is a renowned category of prediction techniques in video coding where the current block is predicted by copying the samples from a similar block that is present somewhere in the already decoded stream of samples. Motion-compensated prediction, intra block copy, template matching prediction etc. are examples. While the displacement information of the similar block is transmitted to the decoder in the bit-stream in the first two approaches, it is derived at the decoder in the last one by repeating the same search algorithm which was carried out at the encoder. Region-based template matching is a recently developed prediction algorithm that is an advanced form of standard template matching. In this method, the reference area is partitioned into multiple regions and the region to be searched for the similar block(s) is conveyed to the decoder in the bit-stream. Further, its final prediction signal is a linear combination of already decoded similar blocks from the given region. It was demonstrated in previous publications that region-based template matching is capable of achieving coding efficiency improvements for intra as well as inter-picture coding with considerably less decoder complexity than conventional template matching. In this paper, a theoretical justification for region-based template matching prediction subject to experimental data is presented. Additionally, the test results of the aforementioned method on the latest H.266/Versatile Video Coding (VVC) test model (version VTM-14.0) yield an average Bjøntegaard-Delta (BD) bit-rate savings of -0.75% using all intra (AI) configuration with 130% encoder run-time and 104% decoder run-time for a particular parameter selection.

Structural conservation of HBV-like capsid proteins over hundreds of millions of years despite the shift from non-enveloped to enveloped life-style.

The discovery of nackednaviruses provided new insight into the evolutionary history of the hepatitis B virus (HBV): The common ancestor of HBV and nackednaviruses was non-enveloped and while HBV acquired an envelope during evolution, nackednaviruses remained non-enveloped. We report the capsid structure of the African cichlid nackednavirus (ACNDV), determined by cryo-EM at 3.7 Å resolution. This enables direct comparison with the known capsid structures of HBV and duck HBV, prototypic representatives of the mammalian and avian lineages of the enveloped Hepadnaviridae, respectively. The sequence identity with HBV is 24% and both the ACNDV capsid protein fold and the capsid architecture are very similar to those of the Hepadnaviridae and HBV in particular. Acquisition of the hepadnaviral envelope was thus not accompanied by a major change in capsid structure. Dynamic residues at the spike tip are tentatively assigned by solid-state NMR, while the C-terminal domain is invisible due to dynamics. Solid-state NMR characterization of the capsid structure reveals few conformational differences between the quasi-equivalent subunits of the ACNDV capsid and an overall higher capsid structural disorder compared to HBV. Despite these differences, the capsids of ACNDV and HBV are structurally highly similar despite the 400 million years since their separation.

Molecular elucidation of drug-induced abnormal assemblies of the hepatitis B virus capsid protein by solid-state NMR.

Hepatitis B virus (HBV) capsid assembly modulators (CAMs) represent a recent class of anti-HBV antivirals. CAMs disturb proper nucleocapsid assembly, by inducing formation of either aberrant assemblies (CAM-A) or of apparently normal but genome-less empty capsids (CAM-E). Classical structural approaches have revealed the CAM binding sites on the capsid protein (Cp), but conformational information on the CAM-induced off-path aberrant assemblies is lacking. Here we show that solid-state NMR can provide such information, including for wild-type full-length Cp183, and we find that in these assemblies, the asymmetric unit comprises a single Cp molecule rather than the four quasi-equivalent conformers typical for the icosahedral T = 4 symmetry of the normal HBV capsids. Furthermore, while in contrast to truncated Cp149, full-length Cp183 assemblies appear, on the mesoscopic level, unaffected by CAM-A, NMR reveals that on the molecular level, Cp183 assemblies are equally aberrant. Finally, we use a eukaryotic cell-free system to reveal how CAMs modulate capsid-RNA interactions and capsid phosphorylation. Our results establish a structural view on assembly modulation of the HBV capsid, and they provide a rationale for recently observed differences between in-cell versus in vitro capsid assembly modulation.

Probing a Hydrogen-π Interaction Involving a Trapped Water Molecule in the Solid State.

The detection and characterization of trapped water molecules in chemical entities and biomacromolecules remains a challenging task for solid materials. We herein present proton-detected solid-state Nuclear Magnetic Resonance (NMR) experiments at 100 kHz magic-angle spinning and at high static magnetic-field strengths (28.2 T) enabling the detection of a single water molecule fixed in the calix[4]arene cavity of a lanthanide complex by a combination of three types of non-covalent interactions. The water proton resonances are detected at a chemical-shift value close to zero ppm, which we further confirm by quantum-chemical calculations. Density Functional Theory calculations pinpoint to the sensitivity of the proton chemical-shift value for hydrogen-π interactions. Our study highlights how proton-detected solid-state NMR is turning into the method-of-choice in probing weak non-covalent interactions driving a whole branch of molecular-recognition events in chemistry and biology.

Disentangling the Effect of Pressure and Mixing on a Mechanochemical Bromination Reaction by Solid-State NMR Spectroscopy.

Mechanical forces, including compressive stresses, have a significant impact on chemical reactions. Besides the preparative opportunities, mechanochemical conditions benefit from the absence of any organic solvent, the possibility of a significant synthetic acceleration and unique reaction pathways. Together with an accurate characterization of ball-milling products, the development of a deeper mechanistic understanding of the occurring transformations at a molecular level is critical for fully grasping the potential of organic mechanosynthesis. We herein studied a bromination of a cyclic sulfoximine in a mixer mill and used solid-state nuclear magnetic resonance (NMR) spectroscopy for structural characterization of the reaction products. Magic-angle spinning (MAS) was applied for elucidating the product mixtures taken from the milling jar without introducing any further post-processing on the sample. Ex situ 13 C-detected NMR spectra of ball-milling products showed the formation of a crystalline solid phase with the regioselective bromination of the S-aryl group of the heterocycle in position 4. Completion is reached in less than 30 minutes as deduced from the NMR spectra. The bromination can also be achieved by magnetic stirring, but then, a longer reaction time is required. Mixing the solid educts in the NMR rotor allows to get in situ insights into the reaction and enables the detection of a reaction intermediate. The pressure alone induced in the rotor by MAS is not sufficient to lead to full conversion and the reaction occurs on slower time scales than in the ball mill, which is crucial for analysing mixtures taken from the milling jar by solid-state NMR. Our data suggest that on top of centrifugal forces, an efficient mixing of the starting materials is required for reaching a complete reaction.